Human progenitor t-cells

ABSTRACT

Human progenitor T cells that are able to successfully engraft a murine thymus and differentiate into mature human T and NK cells are described. The human progenitor T cells have the phenotype CD34+CD7+CD1a−CD5− or CD34+CD7+CD1a−CD5+ and are derived from human hematopoietic stem cells, embryonic stem cells and induced pluripotent stem cells by coculture with cells expressing a Notch receptor ligand (OP9-DL1 or OP9-DL4). Such cells are useful in a variety of applications including immune reconstitution, the treatment of immunodeficiencies and as carriers for genes used in gene therapy.

FIELD

The application relates to progenitor T cells, methods of preparingsame, and all uses of the progenitor T cells including the use of the Tcells to create mature human T-cell populations, to engraft into thymustissue and in therapeutic applications.

BACKGROUND

T cells are the major cellular arm of the immune system that elicitpotent and specific immune responses in vivo against bacterial and viralantigens. Individuals born with severe combined immunodeficiency (SCID)exhibit a complete absence of T cells, while individuals infected withHIV/AIDS or treated for cancer with chemo/radio-therapy exhibit aprofound depletion of T cells. Regardless of whether theimmunodeficiency is congenital or acquired, these individuals arecompromised in their capacity to generate new T cells from incoming bonemarrow-derived stem cells and to mount sufficient immune responsesagainst opportunistic infections. Conversely, individuals with certainautoimmune diseases such as arthritis and diabetes exhibit inappropriateimmune responses against self-tissue due in part to an absence of aparticular kind of T cell termed T-regulatory cells. Thus, the abilityto generate new designer T cells in vitro through the differentiation ofexpanded progenitor cells extracted from a particular individual mayoffer therapeutic benefits in the treatment of many diseases byrestoring T cell numbers and the capacity to maintain and regulate afunctional immune system. An in vitro differentiation system in whichmouse hematopoietic stem cells are induced to differentiate towards a Tcell lineage following a period of coculture with the mouse OP9 bonemarrow stromal cell line that expresses the Notch receptor ligandsDelta-like-1 or -4 has been described, however, the characterizationhuman hematopoietic stem cells using the same system has yet to beelucidated.

Hematopoietic stem cells (HSCs) which give rise to erythroid, myeloid,and lymphoid lineages, can be identified based on the expression of CD34and the absence of lineage specific markers (termed Lin−) (Kawamoto etal., 1997). Human umbilical cord blood (CB) provides a rich source ofHSCs, which are comparable to bone marrow-derived HSCs (Barker andWagner, 2003; de Wynter et al., 1999; Fisher et al., 1990; Galy et al.,1993; Gluckman et al., 1997; Ito et al., 2002; Lewis and Verfaillie,2000; McCune et al., 1991; Sànchez et al., 1993; Wilpshaar et al.,2002). Human T cells differentiate in the thymus via discretedevelopmentally-regulated steps that involve a series of commitmentevents and developmental checkpoints including T cell receptor (TCR)variable (V), diversity (D), and joining (J) gene segment rearrangements[V(D)J], and positive/negative selection of developing thymocytes(Spits, 2002). The earliest intrathymic progenitors express high levelsof CD34 and CD7, do not express CD1a, and are triple-negative (TN) formature T cell markers: CD4, CD8, and CD3 (Galy et al., 1993). Commitmentto the T cell lineage is associated with the expression of CD1a byCD7-expressing pro-thymocytes (Spits, 2002; Spits et al., 2000).

Several studies have implicated the Notch pathway in promoting HSCexpansion, self-renewal (Stier et al., 2002), survival (Deftos andBevan, 2000; Osborne and Miele, 1999), and the induction of T celllineage commitment (MacDonald et al., 2001; Osborne and Miele, 1999;Pear and Radtke, 2003; Radtke et al., 2002; Robey, 1999; von Boehmer,2001). In humans there are four Notch receptors (Ellisen et al., 1991;Lardelli et al., 1994; Milner et al., 1994; Uyttendaele et al., 1996;Weinmaster et al., 1991), which can pair with two serrate like ligands(Jagged 1 & 2) (Lindsell et al., 1995; Luo et al., 1997) or threedelta-like-ligands (DII-1, -3 & -4) (Karanu et al., 2001; Shutter etal., 2000) Notch signaling appears to act at multiple stages of T celldifferentiation (Deftos et al., 2000; Garcí a-Peydró et al., 2003; lzonet al., 2001; Jiang et al., 1998; Robey et al., 1996; Washburn et al.,1997) The strongest evidence for the role of Notch signaling in T celldevelopment comes from gain-of-function and loss-of-function studies(Allman et al., 2002; Izon et al., 2002; MacDonald et al., 2001; Pear etal., 1996; Pui et al., 1999; Radtke et al., 2002; Wilson et al., 2001),in which signaling though Notch-1 was shown to play a crucial role indetermining the B cell versus T cell lineage choice (Pear and Radtke,2003; Radtke et al., 2002).

HSCs express multiple Notch receptors (Milner et al., 1996; Milner etal., 1994) but the expression patterns of the various Notch ligands havebeen reported to be distinct between bone marrow stromal cells (Jones etal., 1998; Karanu et al., 2001; Li et al., 1998; Varnum-Finney et al.,1998; Walker et al., 1999) and thymic epithelial cells (Anderson et al.,2001) Taken together, these results suggest that different Notchreceptors and ligands may control different aspects of hematopoiesisdepending on the microenvironment: allowing for self-renewal in the bonemarrow and influencing cell fate decisions in the thymus (Varnum-Finneyet al., 1998). This led to the hypothesis that bone marrow stromallines, such as OP9 cells (Cho et al., 1999; Kim et al., 2003; Kodama etal., 1994), which support B cell differentiation may do so because theappropriate Notch ligand to induce T cell commitment and differentiationis absent. This hypothesis was tested, and demonstrated that OP9 cells,which do not express DII1, when retrovirally-transduced to express DII-1(OP9-DL1) inhibited the development of B cells and favored thedevelopment of T cells from fetal liver-derived HSCs (Schmitt andZúñiga-Pflücker, 2002) or mouse ESCs (Schmitt et al., 2004). Given thehigh level of homology (90%) between mouse and human DII-1 molecules,and the observation that mouse stromal lines can support thedifferentiation of human HSCs (Bennaceur-Griscelli et al., 2001; Jalecoet al., 2001; Karanu et al., 2001; Rawlings et al., 1995), the inventorssought to determine whether human CB-derived HSCs (CD34⁺CD38⁻) culturedon OP9-DL1 cells could initiate and support T cell differentiation invitro.

T-cells develop within the thymus from bone marrow-derived hematopoieticprogenitors, and follow a series of stage-specific differentiationevents, which are broadly characterized by thedevelopmentally-coordinated expression of CD4 and CD8 (Blom and Spits,2006; Spits, 2002).

The initial stages of human T-cell development include precursors thatexpress the stem cell marker CD34 (Haddad et al., 2006; Hao et al.,2001), which is also present on hematopoietic stem cells (HSCs) and onmultipotent or lineage-specified progenitor cells. Furthermore, severalgroups have established that the most primitive cells in the humanthymus possess multi-lineage potential (Blom et al., 1997; Res et al.,1996; Weerkamp et al., 2006a) as they give rise to T-lineage, as wellas, natural killer (NK), dendritic cells (DCs) and to some extentmyeloid-lineage cells (Blom et al., 1997; La Motte-Mohs et al., 2007).Within the known hierarchy of T-cell development, the earliest precursorsubset is further defined by their lack of CD3, CD4, CD8 and CD1aexpression (Galy et al., 1993; Vanhecke et al., 1995).

While immature stages of T-cell development are typically delineated asCD34⁺CD1a⁻ (most immature) and CD34⁺CD1a⁺ cells, these populationsremain heterogeneous. Of note, CD7 expression is one of the earliestcell surface markers known to appear during T-lymphopoiesis (Haddad etal., 2006; Haynes et al., 1988). Importantly, the transition fromCD34⁺CD7⁺CD1a⁻ to CD34⁺CD7⁺CD1a⁺ by early thymocytes is associated withT-cell commitment, as a small percentage (˜10%) of these cells bearrearrangement at the T-cell receptor β-chain (TCRβ) locus (Blom et al.,1999; Dik et al., 2005). In addition, CD34⁺CD7⁺CD1a⁺ cells appear to beT-lineage restricted, as these cells show low precursor activity towardsnon-T-cell lineages (Spits, 2002). Following this stage, thymocytesprogress to a CD4 immature single positive (CD4ISP) stage, at whichpoint CD4 is expressed in the absence of CD8. Thereafter, a subset ofCD4ISP cells are thought to complete TCR rearrangement leading toβ-selection and differentiation to the CD4⁺CD8⁺ double positive (DP)stage. Finally, following TCRa rearrangement, TCRαβ-expressing DPthymocytes undergo positive and negative selection, and yield CD4⁺CD8⁻and CD4⁻CD8⁺ single positive (SP) T-cells, which emigrate to theperiphery (Vanhecke et al., 1997).

Current understanding of the above-outlined stages has been obtainedfrom analyses of human fetal or adult thymocyte subsets, and byanalyzing T-cell development in vitro using xenogeneic engraftment ofmouse fetal thymus organ cultures (FTOCs) (Fisher et al., 1990; LaMotte-Mohs et al., 2007). While these systems have provided importantinsight into T-cell development, the capacity to evaluate specificprogenitor populations has remained difficult to assess given therequirement of human thymus tissue, and the limited number of progenitorT-cells that can be readily analyzed.

Previous work from the inventors' laboratory established that humanT-lineage differentiation can be induced from umbilical cord-blood(UCB)-derived HSCs cocultured with OP9-DL1 cells (La Motte-Mohs et al.,2005). The inventors showed normal stage-specific expression of variouscell surface molecules, including the generation of immature DPT-lineage cells. However, these studies were not performed usingquantitative clonal analyses, and it was unresolved whether differentUCB CD34⁺ subsets could give rise to T-lineage cells and whetherDelta-like/Notch signals influence the T-progenitor frequency of CD34⁺UCB cells. Additionally, it was unclear whether functional T-cells couldbe generated. Finally, the inventors' initial studies (La Motte-Mohs etal., 2005) showed that during the early stages of HSC/OP9-DL1differentiation a population of cells resembling T-progenitors becameapparent, however the potential of these cells to serve as effectiveT-cell progenitors was not addressed.

SUMMARY

The inventors examined the early stages of human T-cell development invitro, and performed limiting dilution and single-cell assays to addressthe T-cell progenitor frequency of various UCB-derived CD34⁺stem/progenitor subsets. The inventors assessed the effect ofDelta-like/Notch interactions in enhancing T-cell progenitor potentialamong Notch-signaled CD34⁺ subsets. Furthermore, using limiting dilutionthymus-reconstitution approaches, the inventors findings revealed thatdifferent tissue culture-derived T-progenitor subsets vary in theirthymus-engrafting effectiveness, although these cells display a similarpotential to give rise to T-lineage cells when assayed on OP9-DL1 cells.In particular, two distinct subsets, CD34⁺CD7⁺⁺CD5⁻ CD1a⁻ (proT1) andCD34⁺CD7⁺⁺CD5⁺CD1a⁻ (proT2) were analyzed. The inventors also showedthat mature functional T-cells are generated in vitro, and that thesecells upon TCR-stimulation display T-cell effector-function. The pro-Tcells can also give rise to natural killer (NK) cells when cultured withIL-15.

Together, these findings support the use of the progenitor T cells(pro-T cells) for the generation and study of human T-cells and NKcells, and provide support for the use of in vitro-generated pro-Tcells, mature T-cells and NK cells in cell-based immune-reconstitutionapproaches.

Accordingly, one aspect of the present application provides an isolatedprogenitor T cell having the phenotype CD34⁺CD7⁺CD1a⁻. In oneembodiment, the isolated progenitor T cell has the phenotypeCD34⁺CD7⁺CD5⁻CD1a⁻. In another embodiment, the isolated progenitor Tcell has the phenotype CD34⁺CD7⁺CD5⁺CD1a⁻.

In another aspect, the present application provides a pharmaceuticalcomposition comprising an isolated progenitor T cell in admixture with asuitable diluent or carrier.

In another aspect, the present application provides the use of the humanprogenitor T cells in all applications, including preparing mature Tcells, preparing NK cells, engrafting a thymus, immune reconstitution,the treatment of conditions requiring an increase in T cells as carriersfor genes used in gene therapy.

Other features and advantages of the present application will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the application aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the application will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Advantages of the present application can be readily appreciated as thesame becomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings. The following is a brief description of the drawings, whichare presented only for the purposes of further illustrating theapplications and not for the purposes of limiting the same.

FIG. 1. Developmental progression of human T-lineage cells fromCD34⁺CD38^(−/lo) HSCs cultured on OP9-DL1 cells. (A) Flow cytometricanalysis for the cell surface expression of CD34, CD5, CD1a, CD10, CD2,CD4, CD8, CD3 and CD7 from purified human CD34⁺CD38^(−/lo) HSCs prior tococulture with OP9-DL1 cells. (B, C)HSC/OP9-DL1 cocultures wereharvested and analyzed by flow cytometry at the indicated time-pointsfor the expression of the markers as shown. Data are representative ofat least 5 independent cocultures. Numbers in plots indicate percentageof cells within each quadrant.

FIG. 2. Analysis for the presence of T-cell progenitors generated invitro. (A) Flow cytometric analysis for the expression of CD7 and CD45RAfrom HSC/OP9-DL1 cocultures harvested at the indicated time-points,including a day 0 prior to the start of coculture. (B) Flow cytometricanalysis of CD7 and CD34 expression from HSC/OP9-DL1 coculturesharvested at days 4, 6, and 8 (upper row), with CD45RA expression shownfor cells gated as CD34⁺CD7⁺⁺ (lower row). Data are representative from3 independent cocultures. Numbers in plots indicate percentage of cellswithin each quadrant, and RCN=relative cell number.

FIG. 3. Gene expression analysis of CD34⁺CD38^(−/lo) HSCs cultured onOP9-DL1 cells. (A) Temporal kinetics of gene expression by quantitativereal-time Q-PCR analysis from human CD34⁺CD38^(−/lo) HSCs cultured oneither OP9-control or OP9-DL1 cells for 6, 10, 14 and 18 days. (B) Flowcytometric analysis for the cell surface expression of CD7 and CD1a froma day 40 HSC/OP9-DL1 coculture, with CD34 expression shown for cellsgated as CD7⁺CD1a⁻. (C) Gene expression analysis by Q-PCR from thecoculture-derived subsets as indicated in (B), CD34⁺CD7⁺⁺CD1a⁻,CD34⁻CD7⁺⁺CD1a⁻, CD34⁻CD7⁺⁺CD1a⁺, CD34⁻CD7⁺CD1a⁺⁺ see figure key. CD3⁺ Tcells and CD33⁺ myeloid cells were purified from the lineage⁺ fractionof UCB samples and served as controls. Transcript levels for theindicated genes were normalized to human β-actin, and these data arerepresentative of three independent experiments, with the STD error barsshown corresponding to values obtained from triplicate wells within anindividual experiment.

FIG. 4. Characterization of CD8⁺ T cells generated in vitro. (A) Flowcytometric analysis for the expression of CD8 and CD4 from humanUCB-derived HSCs cultured on OP9-DL1 cells for 65 days. CD8⁺CD4⁻ singlepositive (SP) cells were gated, as indicated, and analyzed for theexpression of CD27 and CD3, with CD1a expression shown for cells gatedas CD3⁺CD27⁻ or CD27⁺ (shaded and clear histograms, respectively). (B-D)Day 60-70 HSC/OP9-DL1 coculture-derived CD8 SP T cells were purified asshown, CD8⁺CD4⁻ and CD3⁺, and stimulated anti-CD3/CD28 mAbs for 5 days.Flow cytometric analyses of stimulated (S) or control (non-stimulated,NS) CD8⁺CD3⁺ cells (clear and shaded histograms, respectively) for theexpression of CD45RO, CD27, MHC-class II and CD38 (B); CFSE levels andCD25 (lower row), with cell size measured by Forward-light Scatter (FSC)intensity (C); and, CD3 and intracellular Granzyme B (D) are shown. (E)Human IFNγ levels from culture supernatants derived from theabove-outlined experiment (B) were determined by ELISA. Statisticalsignificant was measured by unpaired t-Test. * (p<0.005) 2 μg/mlanti-CD3/CD28 stimulated group versus non-stimulated control. **(p<0.0005) 10 μg/ml anti-CD3/CD28 versus non-stimulated control. Dataare representative of at least 3 independent experiments, with theexception of the data from the 10 μg/ml stimulations, which are derivedfrom 2 independent experiments.

FIG. 5. Analysis of engraftment and differentiation by in vitro-derivedprogenitor T cell subsets in FTOC. UCB CD34⁺CD38^(−/lo) HSCs weredifferentiated for 13 days on OP9-DL1 cells and CD34⁺CD7⁺⁺CD5⁻ (proT1)and CD34⁺CD7⁺⁺CD5⁺ (proT2) subsets were sorted by flow cytometry asindicated in (A), and placed into FTOC (B) or placed back onto OP9-DL1cells (C) for 19 days. Cells were harvested and analyzed for cellsurface expression of CD45, CD7, CD34, CD5, CD1a, CD8, and CD4. Data arerepresentative of 3 independent experiments in which 1.5×10⁴ sorted proTsubsets were placed either into fetal thymus lobe-pairs or in wellscontaining OP9-DL1 cells.

FIG. 6. Gene expression analysis of CD34⁺CD7⁺⁺CD5⁻ and CD34⁺CD7⁺⁺CD5⁺proT cell subsets. (A) Q-PCR analysis of CD34⁺CD7⁺⁺CD5⁻ (proT1) andCD34⁺CD7⁺⁺CD5⁺ (proT2) cells purified by flow cytometric cell sortingfrom a day 14 HSC/OP9-DL1 coculture. Thymocytes obtained from the Lin⁻fraction of a human post-natal thymus (PNT) served as a control sample.Transcript levels for the indicated genes [Ccr9 (CD199), Selplg1(PSGL-1, CD162), Itga2 (α2, CD49b), Itga4 (α4, CD49d), Itga5 (α5,CD49e), and Itgb1 (β1, CD29)] were normalized to human β-actin. Thesedata are representative of 3 independent experiments, with the STD errorbars shown corresponding to values obtained from triplicate wells withinan individual experiment. (B) Flow cytometric analysis for cell surfaceexpression of CD49d on gated CD34⁺CD7⁺⁺CD5⁻ (proT1, open) andCD34⁺CD7⁺⁺CD5⁺ (proT2, shaded) cells from a day 11 HSC/OP9-DL1coculture.

FIG. 7. Analysis of total cellularity from HSC/OP9-DL1 cocultures atdifferent time-points. Human CD34⁺CD38^(−/lo) HSCs (1×10⁴) from fourseparate cord bloods were placed into a well of a 6-well platecontaining OP9-DL1 cells. Cellularity was determined at the indicatedtime-points by counting cells under the microscope with a hemocytometerbased on trypan blue exclusion, and fold expansion determined by thecellularity obtained at the indicated day divided by the initial inputof HSCs.

FIG. 8. Characterization of the UCB-derived CD34⁺ subsets used for theprogenitor frequency determination in limiting dilution assay.Lineage-depleted UCB cells were gated to exclude CD7-expressing cells,sorted into CD34⁺CD38⁻, CD34⁺CD38^(lo), and CD34⁺CD38^(+/hi) subsets,plated onto OP9-DL1 cells, and cultured for 11 days. Results from thelimiting dilution assay are shown in Table I.

FIG. 9. Gene expression analysis of CD34⁺CD7⁺⁺CD5⁻ and CD34⁺CD7⁺⁺ CD5⁺subsets. Q-PCR analysis for the expression of Cebpα and Gata-2 fromCD34⁺CD7⁺⁺CD5⁻ (proT1) and CD34⁺CD7⁺⁺CD5⁺ (proT2) cells sorted from aday 14 HSC/OP9-DL1 coculture. Lin⁻ thymocytes obtained from human PNT orCD33⁺ myeloid cells obtained from the Lin⁺ fraction of UCB served ascontrols. Transcript levels for the indicated genes were normalized tohuman β-actin. These data are representative of 3 independentexperiments, with the STD error bars shown corresponding to valuesobtained from triplicate wells within an individual experiment.

FIG. 10. Analysis of long-term HSC/OP9-DL1 cocultures. CD34⁺CD38^(−/lo)cells cultured on OP9-DL1 cells for 40, 80 and 120 days were analyzed byflow cytometry for the expression of CD7, CD34, CD8 and CD4. These dataare representative of at least 5 independent experiments.

FIG. 11. (Top panel) Flow cytometric analysis and gating on lymphocytesby FSC and SSC gating in thymus of indicated mice. (Lower panel)Following live lymphocyte gating, cells were analyzed based on SSC andhuman CD45 staining. Numbers in plots indicate percentage of cellswithin each quadrant.

FIG. 12. CD45⁺-gated flow cytometric analysis for the expression ofCD34, CD7, CD5 and CD1a from thymuses of two reconstituted mice. Numbersin plots indicate percentage of cells within each quadrant.

FIG. 13. CD45+-gated flow cytometric analysis for the expression of CD4,CD8, and CD3 cell surface expression from thymuses of two reconstitutedmice. Numbers in plots indicate percentage of cells within eachquadrant.

FIG. 14. Following live lymphocyte gating, cells were analyzed based onSSC and human CD45 staining from thymus of indicated mice. Numbers inplots indicate percentage of cells within each quadrant.

FIG. 15. CD45⁺-gated flow cytometric analysis for the expression of CD4,CD8, and CD3 cell surface expression from the thymus of anHSC-reconstituted (top) and proT reconstituted (lower) mouse. Inset plotshows CD4 and CD8 expression on CD3^(hi) gated cells. Numbers in plotsindicate percentage of cells within each quadrant. NOD/SCID γc^(−/−)figure legend

FIG. 16. Flow cytometric analysis of thymuses from NOD/SCID γc^(−/−)mice receiving either CD34⁺ HSCs or CD34⁺CD7⁺ progenitor T cells.Following live lymphocyte gating, cells were analyzed for the expressionof CD7, CD5, CD1a, CD4 and CD8 expression after SSC and human CD45gating. Numbers in plots indicate percentage of cells within eachquadrant.

FIG. 17. Flow cytometric analysis of human HSCs induced to differentiateon OP9-control, OP9-DL1 and OP9-DL4 cells. Following live lymphocytegating, day 24 cocultured cells were analyzed for the expression of CD4,CD8, and pre-Tα expression. Numbers in plots indicate percentage ofcells within each quadrant.

FIG. 18. Flow cytometric analysis of human HSCs induced to differentiateon OP9-control, OP9-DL1 and OP9-DL4 cells. Following live lymphocytegating, day 24 cocultured cells were analyzed for the expression of CD7,CD1a, and CD5 (right 3 columns) expression. Gated on T cell populations(left column) corresponding to (A) CD7⁺CD1a⁺⁺ (more mature T cells), (B)CD7⁺⁺CD1a⁺ (committed T cells), and (C) CD7⁺⁺CD1a⁻ (progenitor T) andwere examined for CD5 (right 3 columns) expression in each of thecorresponding populations labeled as A, B or C. Numbers in plotsindicate percentage of cells within each gated population.

FIG. 19. Flow cytometric analysis of human HSCs induced to differentiatetowards the T lineage upon coculture with OP9-DL1 and OP9-DL4 cells.Following live lymphocyte gating, day 40 cocultured cells were analyzedfor the expression of CD7, CD1a, TCR-αβ, and TCR-γδ expression. Gated onT cell populations (top row) corresponding to (A) CD7⁺CD1a⁺⁺ (moremature T cells), (B) CD7⁺⁺CD1a⁺ (committed T cells), and (C) CD7⁺⁺CD1a⁻(progenitor T) were examined for TCR-αβ (left panel, bottom 3 rows), andTCR-γδ (right panel, bottom 3 rows) expression in each of thecorresponding populations labeled as A, B or C. Numbers in plotsindicate percentage of cells of positive cells within each gatedpopulation.

FIG. 20. Flow cytometric analysis of human HSCs induced to differentiateon OP9-control, OP9-DL1 and OP9-DL4 cells. Following live lymphocytegating, day 40 cocultured cells were analyzed for the expression of CD3,V-beta (V_(β))-3,5,8,23 (right panel). Corresponding isotype controlsare shown (left panel). Numbers in plots indicate percentage of cellswithin each quadrant.

FIG. 21. ProT1 cells directly give rise to proT2 cells in vitro. Flowcytometric analysis of CD34⁺CD7⁺⁺CD5⁻ (proT1) and CD34⁺CD7⁺⁺CD5⁺ (proT2)cells. Left-side panels, sorted in vitro-generated-proT1 cells wereplaced onto OP9-DL1 cells and the acquisition of cell surface CD5 wasexamined at the indicated time points. Right-side panels, as a control,proT2 were sorted and re-cultured on OP9-DL1 cells. All plots shown weregated for CD34⁺CD7⁺⁺ expression for the analysis.

FIG. 22. Natural killer (NK) cell differentiation potential ofCD34⁺CD7⁺⁺CD5⁻ (proT1) and CD34⁺CD7⁺⁺CD5⁺ (proT2) cells. Sorted proT1and proT2 cells from a day 10 HSC/OP9-DL1 coculture were placed ontoOP9-control cells supplemented with rhlL-15 (5 ng/mL); or proT1 andproT2 cells were placed back onto OP9-DL1 cells. The expression of theNK cell lineage marker CD56 was examined after 12 days of culture.

FIG. 23. Human proT2 cells effectively engraft the thymus ofimmunodeficient mice and facilitate thymic engraftment of UCB-derivedHSCs. Analysis of thymus engraftment and differentiation of HSCscoinjected with in vitro-derived proT2 cells into immunodeficient mice.Human UCB CD34⁺CD38^(−/lo) (HLA-A2⁻) cells were differentiated onOP9-DL1 cells for 10-12 days, and CD34⁺CD7⁺⁺CD5⁺ (proT2) cells weresorted by flow cytometry. On the same day that proT2 cells were sorted,human UCB CD34⁺CD38^(−/lo) (HLA-A2⁺) cells were also sorted. Irradiated(130 cGy) neonatal NOD/SCID/γc^(null) mice from the same litter wereinjected intrahepatically with 3.5×10⁴ HSCs; 2.5×10⁵ proT2 cells; or3.5×10⁴ HSCs mixed with 2.5×10⁵ proT2 cells. Bone marrow (BM), spleenand thymuses were harvested 6 weeks after injection, single cellsuspensions were obtained and analyzed by flow cytometry. The analysiswas performed by gating for human CD45⁺ cells and donor cell type(HSC-derived-A2⁺, or proT2-derived-AZ), based on the absence(proT2-derived) or presence (HSC-derived) of HLA-A2 expression on CD45⁺cells. Flow cytometric analysis for cell surface expression of CD45 andHLA-A2 of (A) BM and (B) spleens from HSC only, HSC+proT2, and proT2only, treated mice are shown. The lower rows show CD19 and CD33 cellsurface staining on CD45⁺ HLA-A2⁺ (second row; HSC-derived) and CD45⁺HLA-A2⁻ (third row; proT2-derived) gated cells. (C) Flow cytometricanalysis for cell surface expression of CD45 and HLA-A2 of thymuses fromHSC only, HSC+proT2, and proT2 only, treated mice are shown. The upperrow displays CD45 and HLA-A2 cell surface staining. The lower rows showCD45 and CD3 cell surface staining on CD45⁺ HLA-A2⁺ (second row;HSC-derived) and CD45^(+HLA-A)2⁻ (third row; proT2-derived) gated cells.(D) Flow cytometric analysis of thymuses from HSC+proT2 coinjected mice.The upper row displays CD45 and HLA-A2 cell surface staining. The lowerrows show CD8 and CD4 cell surface staining on CD45⁺ HLA-A2⁺ (secondrow; HSC-derived) and CD45⁺ HLA-A2⁻ (third row; proT2-derived) gatedcells.

FIG. 24. Human ESCs and human iPSCs can generate early human T-lineageprogenitor cells upon coculture with OP9-DL1 or OP9-DL4 cells. (A) Usingthe two-stage protocol method (see text for details), CD34⁺⁺ cellssorted from embryoid bodies, were placed onto OP9-DL1 cells and examinedfor cell surface expression of CD5 and CD7 after 20 days of culture;and, (B) human iPSCs aggregated to form embryoid bodies weresequentially induced to differentiate towards the hematopoietic lineage,sorted CD34⁺⁺ cells were placed onto OP9-DL4 cells and analyzed for theexpression of CD7 and CD5 by flow cytometry as indicated.

DETAILED DESCRIPTION

T cell development is known to follow a defined set of stage-specificdifferentiation steps. However, the molecular and cellular eventsoccurring at early stages of human T-cell development have notpreviously been fully elucidated. To address this, human umbilical cordblood (UCB)-derived hematopoietic stem cells (HSCs) were induced todifferentiate to the T-lineage by coculture with OP9-DL1 cells. Adevelopmental program was revealed that is highlighted by an early,sequential and temporally discrete expression of CD34, CD7, CD45RA, CD5,CD1a, CD2, and CD4. Quantitative clonal analyses demonstrated thatCD34⁺CD38⁻ and CD34⁺CD38^(lo) subsets of UCB cells contain a similarlyhigh T-cell progenitor frequency of 1 in 4 cells, while the frequency inCD34⁺CD38^(+/hi) cells was 5-fold lower. To address whetherDelta-like/Notch-induced signals can affect the T-cell progenitorfrequency of UCB CD34⁺CD38^(−/lo) cells differentiated on OP9-DL1 cells,two distinct subsets, CD34⁺CD7⁺⁺CD5⁻CD1a⁻ (proT1) andCD34⁺CD7⁺⁺CD5⁺CD1a⁻ (proT2) were analyzed, and both subsets showed a2-fold increase in frequency. The inventors established that theseprogenitor subsets are able to successfully engraft a mouse thymus anddifferentiate into CD4 and CD8 human T-cells in vitro. Surprisingly, thein vitro-generated proT2 cells showed a 3-fold enhancedthymus-engrafting capacity in vitro than the more immature proT1progenitor subset. The proT2 cells also showed an almost 3-foldenhancement in thymus-engrafting capacity in vivo than the proT1 cells.Further analysis of these subsets showed that proT2 cells express higherlevels of CCR9, PSGL-1 and key integrins than proT1 cells, which mayallow for the enhanced engrafting ability. Moreover, the inventors alsodemonstrate that human HSC/OP9-DL1 cocultures support the generation ofmature functional αβ-T cell receptor/CD3⁺CD8 T-cells. Further, in thepresence of IL-15, the proT cells can generate natural killer (NK)cells. In addition to human hematopoietic stem cells, the proT cells canalso be generated from embryonic stem cells and induced pluripotent stemcells. Lastly, the inventors have extended the in vitro studies thatdemonstrate thymus reconstitution ability within FTOC, towards in vivomodels that show human thymic reconstitution within immodeficientstrains of mice through intrahepatic injection of progenitor-T cells.Taken together, the generation and identification of defined invitro-generated T-progenitor subsets, which are readily differentiateinto functionally mature T-cells and NK cells in vitro and engraft bothin FTOC (in vitro) and immunodeficient mice (in vivo), may offerimportant avenues to improve cellular based immune-reconstitutionapproaches for the treatment of immunodeficiencies.

I. Progenitor T Cells

Generally, the present application provides isolated progenitor T-cells.

In one aspect, the present application provides an isolated humanprogenitor T cell having the phenotype CD34⁺CD7⁺CD1a⁻. In oneembodiment, the isolated progenitor T cell has the phenotypeCD34⁺CD7⁺CD5⁻CD1a⁻ (pro-T1). In another embodiment, the isolatedprogenitor T cell has the phenotype CD34⁺CD7⁺CD5⁺CD1a⁻ (pro-T2). In aspecific embodiment, the pro-T2 cells express CCR9, PSGL-1 andintegrins.

The term “isolated” as used herein means that the progenitor cell hasbeen separated or purified from cellular or biological material foundwith the cells in their native environment. It thus distinguishes thecells from how they exist in nature.

The term “a cell” or “the cell” includes a plurality of cells.

The term “progenitor T cell’ or “pro-T cell” as used herein means a Tcell that is capable of maturing into a mature T cell or lymphocyte. Amature T cell includes CD4⁺ and CD8⁺ T cells. A lymphocyte includesCD56+NK cells.

The progenitor T cell is preferably human and derived from a stem cellor progenitor cell. Stem or progenitor cells may be obtained from anysuitable source, including, without limitation, umbilical cord, blood,embryos, embryonic tissue, fetal tissue, bone marrow and blood. In oneembodiment, the stem or progenitor cell is a hematopoietic stem orprogenitor cell. In another embodiment, the stem cell is an embryonicstem cell. In a further embodiment, the stem cell is an inducedpluripotent stem cell. For therapeutic applications, the stem cells orprogenitor cells used to generate the progenitor T cells may bepreferably obtained from the patient to be treated.

Progenitor T cells may be isolated from the stem or progenitor cells bytechniques known in the art. Typically, a sample containing the cells isfirst depleted with non-stem cells or mature cells.

Negative and positive selection methods known in the art may be used forenrichment of the progenitor cells. For example, cells can be sortedbased on cell surface antigens using a fluorescence activated cellsorter, or magnetic beads which bind cells with certain cell surfaceantigens. Negative selection columns can be used to remove cellsexpressing lineage specific surface antigens.

In an embodiment, a sample containing stem or progenitor cells isseparated into lineage-negative (Lin⁻) and lineage position (Lin⁺)fractions. The Lin− fraction can be sorted for CD34⁺ cells.

The enriched progenitor cells or stem cells are cultured under suitableconditions to generate pro-T cells. Preferably, the cells are culturedin the presence of one or more Notch ligand for a sufficient time toform pro-T cells. More preferably, the stem cells are cultured in thepresence cells expressing a Notch ligand. This is described in greaterdetail in US-2004-0171148-A1 which is incorporated herein by reference.

In an embodiment, the progenitor cells or stem cells are cultured in a 6cm or 10 cm tissue culture-treated dish with a Notch Ligand CellPreparation. For example, the concentration of hematopoietic progenitorcells or embryonic stem cells in the culture is between 1-10⁹,preferably 1×10² to 1×10⁶, more preferably 1×10³ to 1×10⁴. In aparticular embodiment, hematopoietic progenitor cells or embryonic stemcells (about 1-5×10⁴ cells) are cultured on a monolayer of OP9 cellsexpressing Delta-like-1 or Delta-like 4.

One or more positive cytokines that promote commitment anddifferentiation of pro-T cells may also be added to the culture. Thecytokines may be human in origin, or may be derived from other species.The concentration of a cytokine in a culture is typically about 1-10ng/ml. The following are representative examples of cytokines that maybe employed in the present application: all members of the fibroblastgrowth factor (FGF) family including FGF-4 and FGF-2, Flt-3-ligand, andinterleukin-7 (IL-7). Preferably the cytokines used herein areFlt-3-ligand and IL-7. The cytokines may be used in combination withequal molar or greater amounts of a glycosaminoglycan such as heparinsulfate. The cytokines are commercially available or can be produced byrecombinant DNA techniques and purified to various degrees. Some of thecytokines may be purified from culture media of cell lines by standardbiochemical techniques.

The progenitor cells and stem cells may be cultured in culture mediumcomprising conditioned medium, non-conditioned medium, or embryonic stemcell medium. Examples of suitable conditioned medium include IMDM, DMEM,or αMEM, conditioned with embryonic fibroblast cells (e.g. humanembryonic fibroblast cells or mouse embryonic fibroblast cells), orequivalent medium. Examples of suitable non-conditioned medium includeIscove's Modified Delbecco's Medium (IMDM), DMEM, or αMEM, or equivalentmedium. The culture medium may comprise serum (e.g. bovine serum, fetalbovine serum, calf bovine serum, horse serum, human serum, or anartificial serum substitute) or it may be serum free.

The culture conditions entail culturing the progenitor cells or stemcells for a sufficient period of time so that cells in the preparationform pro-T cells. The cells are maintained in culture generally for 4-50days, preferably 5 to 20 days. It will be appreciated that the cells maybe maintained for the appropriate amount of time required to achieve thedesired cellular composition.

Accordingly, the present application provides a method of generating apro-T cell comprising (a) culturing a sample comprising stem cells orprogenitor cells with cells that express a Notch ligand and (b)isolating pro-T cells. The cells expressing a Notch ligand arepreferably OP9 cells expressing DL1 or DL4. The pro-T cells may becharacterized by the phenotype CD34⁺CD7⁺CD1a⁻.

The methods of the present application allow the generation of largenumbers of pro-T cells. The pro-T cells exhibit, or have the potentialto differentiate into cells that exhibit morphological, physiological,functional, and/or immunological features of T cells. The generation oflarge numbers of pro-T cells with the ability to form mature T cellsmakes them highly useful in cell therapy.

In another embodiment, the progenitor T-cell is obtained by co-culturingstem cells such as HSC with OP9-DL1 cells or OP9-DL4 cells,fractionating the cells and collecting the cells of a desired phenotype.The fractionation step may involve any suitable cell separationtechnique known in the art such as (density gradient, ferromagneticbeads cytometry and fluorescence activated call sorting.) Bioreactors(matrices). In particular, the cells cultured on the OP9-DL1 or OP9-DL4cells may be further fractionated into CD5⁺ (pro-T2) and CD5⁻ (pro-T1)subsets. The pro-T2 subset may be preferably used for T-cellengraftment.

In another embodiment, the progenitor T cells may be used to generate NKcells when cultured under appropriate conditions. Appropriate conditionsto generate NK cells include culturing the pro-T cells with cytokinessuch as IL-15 or IL-2. The pro-T cells can also be cultured stromalcells such as OP-9 cells. Accordingly, the present application providesa method of generating natural killer (NK) cells comprising a) culturingan isolated progenitor cell with IL-15 and b) isolating NK cells. The NKcells may be characterized by the phenotype CD56⁺.

II. Pharmaceutical Compositions

In another aspect, the present application provides a pharmaceuticalcomposition comprising isolated pro T-cells and a pharmaceuticallyacceptable diluent or carrier.

Suitable diluents and carriers are described, for example, inRemington's Pharmaceutical Sciences. On this basis, the compositionsinclude, albeit not exclusively, solutions of the pro-T cells inassociation with one or more pharmaceutically acceptable vehicles ordiluents, and contained in buffered solutions with a suitable pH andiso-osmotic with the physiological fluids.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which may further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that may be present in such compositions include water,surfactants (such as Tween™), alcohols, polyols, glycerin and vegetableoils, for example. Extemporaneous injection solutions and suspensionsmay be prepared from sterile powders, granules, tablets, or concentratedsolutions or suspensions. The composition may be supplied, for examplebut not by way of limitation, as a lyophilized powder which isreconstituted with sterile water or saline prior to administration tothe patient.

Suitable pharmaceutically acceptable carriers include essentiallychemically inert and nontoxic compositions that do not interfere withthe effectiveness of the biological activity of the pharmaceuticalcomposition. Examples of suitable pharmaceutical carriers include, butare not limited to, water, saline solutions, glycerol solutions,ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride(DOTMA), diolesyl-phosphotidyl-ethanolamine (DOPE), and liposomes. Suchcompositions should contain a therapeutically effective amount of thecompound, together with a suitable amount of carrier so as to providethe form for direct administration to the patient.

The compositions of the application can be administered for example, byparenteral, intravenous, subcutaneous, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol or oraladministration. For parenteral administration, solutions of the pro-Tcells described herein can be prepared in water suitably mixed with asurfactant such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, DMSO and mixturesthereof with or without alcohol, and in oils. Under ordinary conditionsof storage and use, these preparations contain a preservative to preventthe growth of microorganisms. A person skilled in the art would know howto prepare suitable formulations.

Preferably the pre T-cells are present in an amount effective fortreating a disease state in a mammalian need thereof. In one embodimentthe pre T-cell is present in an amount effective to enhancehematopoietic progenitor cell engraftment in a mammal in need thereof.Optionally, the composition further comprises pre T-cells, or tissue fortransplantation. In one embodiment the tissue comprises a thymus. Inanother embodiment the tissue comprises an organ.

III. Applications

The present application includes the use of pro-T cells in any and allapplications.

A. Genetic Modification

Pro-T cells generated using the methods of the application may begenetically modified (transduced or transfected) either in nature or bygenetic engineering techniques in vivo or in vitro. Cells can bemodified by introducing mutations into genes in the cells or byintroducing transgenes into the cells. Insertion or deletion mutationsmay be introduced in a cell using standard techniques. A gene encoding aselectable marker may also be integrated into the cells.

An aspect of the present application relates to pro-T cells that aregenetically engineered in such a manner that the cells or cells derivedtherefrom produce, in vitro or in vivo, polypeptides, hormones andproteins not normally produced in the cells in biologically significantamounts, or produced in small amounts but in situations in whichregulatory expression would lead to a therapeutic benefit. For example,the cells could be engineered with a gene that expresses insulin atlevels compatible with normal injected doses, or with a gene that canmake up for a deficiency or abnormality of a gene causing a disease.Alternatively the cells could be modified such that a protein normallyexpressed will be expressed at much lower levels. These products wouldthen be secreted into the surrounding media or purified from the cells.The cells formed in this way can serve as continuous short term or longterm production systems of the expressed substance.

Thus, in accordance with this aspect of the application, pro-T cellsgenerated using the methods of the application can be modified withgenetic material of interest. The modified cells can be cultured invitro under suitable conditions so that they are able to express theproduct of the gene expression or secrete the expression product. Thesemodified cells can be administered so that the expressed product willhave a beneficial effect.

In a further embodiment, transduced pro-T cells (with the potential toform mature T cells) can be induced in vivo to differentiate into Tcells that will express the gene product. For example, the transducedcells may be administered to induce production of T cells having thetransduced gene. The cells may be administered in a mixture with othercells or separately and may be delivered to a targeted area. The cellscan be introduced intravenously and home to a targeted area.Alternatively, the cells may be used alone and caused to differentiatein vivo.

Thus, genes can be introduced into cells that are then injected into arecipient where the expression of the gene will have a therapeuticeffect. For example, an insulin gene may be introduced into the cells toprovide a constant therapeutic dose of insulin in the bone marrow andperipheral blood.

The technology may be used to produce additional copies of essentialgenes to allow augmented expression by T cells of certain gene productsin vivo. These genes can be, for example, hormones, matrix proteins,cell membrane proteins, and cytokines.

In a specific embodiment, the pro-T cells are engineered to recognize anantigen such as a tumor antigen, a viral antigen or a bacterial antigen.As such the immune response to the target antigen will be augmented byadministering antigen specific progenitor T cells.

B. Therapeutic Applications

The ability to generate in vitro-derived human progenitor T cells and totest their safety in human/mouse immune engraftment models, opensavenues for cellular based approaches for treating immune-relateddisorders of the T lineage (Legrand et al., 2006; van den Brink et al.,2004). T cells are the major effector arm of the adaptive immune systemin recognizing and eliminating viral and bacterial pathogens. In certainrare blood cancers such as T cell acute lymphoblastic leukemia (T-ALL),T cells proliferate crowding out healthy immune cells and perturbingnormal immune function (Ferrando et al., 2002; Weng et al., 2004).Although chemotherapy can often impart therapeutic benefits in cancerpatients, it often can lead to immuno-deficiency and susceptibility toopportunistic infections. Opportunistic infections also pose a seriousconcern in AIDS patients whose CD4⁺ T cells have been depleted followinginfection with HIV. While immunodeficiency remains a serious concern inHIV/AIDS and cancer, immune-hypereactivity is equally problematic inautoimmune disease where T cells that lack proper regulatory control,make immune responses to self-tissue.

Accordingly, the present application includes a method of treating ananimal having a condition requiring an increase in the number of T cellscomprising administering an effective amount of a progenitor T cell toan animal in need thereof.

As used herein, the phrase “effective amount” or “therapeuticallyeffective amount” means an amount effective, at dosages and for periodsof time necessary to achieve the desired result. Effective amounts mayvary according to factors such as the disease state, age, sex, weight ofthe animal. The amount of a given cell preparation that will correspondto such an amount will vary depending upon various factors. Such as thepharmaceutical formulation, the route of administration, the type ofdisease or disorder, the identity of the subject or host being treated,and the like, but can nevertheless be routinely determined by oneskilled in the art. An “effective amount” will preferably be an amounteffective for the progenitor T cells to engraft the subject beingtreated.

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e. not worsening) state of disease,preventing spread of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, diminishment of thereoccurrence of disease, and remission (whether partial or total),whether detectable or undetectable. “Treating” and “Treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. “Treating” and “treatment” as used herein alsoinclude prophylactic treatment.

The term “animal” as used herein means any member of the animal kingdomand is preferably a human.

A “condition requiring an increase in number of T cells” includes anycondition wherein T cell levels are reduced as compared to a healthyanimal, including, without limitation, immunodeficiency, cancer, geneticdiseases, infectious diseases and autoimmunity, some of which aredescribed in detail below.

(i) Cancer

In 2005, nearly 128,000 individuals were diagnosed with myeloma,lymphoma and leukemia in North America (US & Canada). Followingaggressive myeloablative-chemo/radiotherapy of these blood cancers,these individuals may become immunodeficient and require stem celltransplantation to replace or restore their immune system. Indeed, everyyear in North America 9,000 individuals undergo stem celltransplantation. Although, HSCs may be obtained from bone marrow,GM-CSF-mobilized peripheral blood, or cord blood, several clinicalchallenges present themselves in most stem cell transplantations: fromfinding a suitably major-histocompatible matched donor, to preventingGvHD, to successful engraftment of a donor immune system onto the host(Socie, 2005). Most immune cells recover quickly followingtransplantation, but T cells take the most time (˜2 years) to recover interms of cell numbers and function (Petropoulos and Chan, 2005). This isperhaps dictated by the broad repertoire of TCRs required to cover therange of environmental and pathogenic antigens that an individual willbe exposed to. Until that broad repertoire is re-established, gaps mayexist that permit the emergence of opportunistic infections.

Accordingly, the present application provides a method of treating orpreventing cancer comprising administering an effective amount of aprogenitor T cell to an animal in need thereof.

In one embodiment, the pro-T cells have been genetically engineered torecognize tumor specific antigens. For example, progenitor T cells couldbe manufactured to recognize tumor-specific antigens found in certainbreast cancers as well as Burkitt's lymphoma, neuroblastoma, malignantmelanoma, osteosarcoma, and renal cell carcinoma (Renkvist et al.,2001). One example of this genetic approach utilizing CD8⁺ Wilms' tumor(WT1) gene-specific cytotoxic T-lymphocyte clones for the treatment ofChronic Myeloid Leukemia (CML) or Acute Lymphoblastic Leukemia (ALL).Thus, progenitor T cell transplantation could be used as an adjuvanttherapy with stem cell transplantation to quickly reconstitute the Tcell compartment in patients with terminal illness or specificallytarget cancer cells for destruction (van den Brink et al., 2004).

(ii) HIV/AIDS:

Acquired Immunodeficiency Syndrome (AIDS), which follows the infectionwith the Human Immunodeficiency Virus (HIV), is characterized by achronic decline in the number of CD4 helper T cells. The CD4 T cell is acritical immune or white-blood cell that helps to maintain the functionof “killer” CD8 cytotoxic T cells, which lyse virus-infected cells(Grossman et al., 2006). AIDS has become a global pandemic with anestimated 38 million people living with the disease worldwide and 1.6million cases in North America alone. Current treatment regimensincluding the highly active anti-retroviral therapy (HAART), acombination of several anti-HIV drugs [i.e.: Viramune (nevirapine),Rescriptor (delavirdine), Invirase (saquinavir), and Norvir(ritonavir)], have been effective in reducing viral load and extendingthe life-span of HIV-infected individuals but have proven difficult toimplement/achieve/maintain over long-periods of times for a variety orreasons (i.e.: toxicity, financial burden, government apathy, andevolving resistance of HIV to these drugs). Indeed, HAART is often givenin cycles with ‘vacations’/break periods to allow the patient to recoverfrom anti-viral drug induced toxicity. As a result there is continuedinterest to find more efficacious drugs and/or cellular based therapies(i.e. vaccines or stem cell approaches) that keep pace with the evolvingresistance of HIV and would augment or replace current treatmentregimens to restore or maintain T cell numbers.

In the case of HIV/AIDS, the value of the present application may be theability to create large numbers of in vitro-generated progenitor T cellsthat would offer therapeutic benefits to individuals that HAART hasfailed or that have gone off HAART due to drug toxicity. One advantageof a progenitor T cell based therapy would be minimal toxicity andside-effects of these cells compared to anti-retrovirals. Given, thatfew treatment options are available to this subpopulation of individualsthat have failed HAART, the use of progenitor T cells could be a viableoption. Although these progenitor T cells and their CD4⁺ progeny wouldagain be subject to HIV infection in vivo and require multipletreatments, the capacity to expand non-infected cells in vitro andrestore T cell numbers in vivo may help to restore immune function andlimit the emergence of opportunistic infections for some time duringperiods of planned HAART ‘vacation’ or failure. This presents two futureextensions of this technology for therapeutic potential. First, theOP9-DL1 coculture system may have therapeutic potential as an adjuvanttherapy in combination with HAART, or as a stand-alone therapy whenHAART is periodically discontinued. As with the case of cancer, theOP9-DL1 coculture system lends itself towards emerging geneticapproaches to create designer T cells resistant to HIV infection. Oneexample of such an innovative approach would be the expression of themutant form of the chemokine co-receptor CCR5 that blocks viralinfection (Markovic, 2006; Samson et al., 1996) in progenitor T cells.Such an approach would offer a novel means to treat HIV/AIDS bypreventing HIV infection and thus maintaining T cell numbers and T cellfunction and is no longer far-fetched as several clinical trials havebeen approved for the treatment of HIV/AIDS using genetically modifiedmature-CD4⁺ T cells and CD34⁺ HSCs, but not-progenitor T cells.

Accordingly, the present application provides a method of treating orpreventing an immunodeficiency comprising administering an effectiveamount of a progenitor T cell to an animal in need thereof. In oneembodiment, the immunodeficiency is HIV/AIDS.

(iii) Autoimmunity

Traditionally, tolerance was thought to be established centrally in thethymus to self-antigen presented by thymic cells and blood-borneself-antigens, while T cells with specificity towards tissue-specificantigens underwent tolerance induction in the periphery (Kyewski andDerbinski, 2004). The recent observation that thymic epithelial cellsthat express the AIRE gene can promote the promiscuous expression oftissue-restricted antigens has yielded new insights for howself-tolerance is maintained and broken (Kyewski and Derbinski, 2004).Autoimmune diseases result from the dysregulation or breakdown of theprocesses that maintain self-tolerance in the periphery. Manyinvestigators have demonstrated that a population of T cells withregulatory activity (T_(Reg)) can suppress pathological immune responsesin murine models of autoimmune disease, transplantation and GvHD(Chatenoud et al., 2001) suggesting that these cells could be utilizedtherapeutically to treat human autoimmune disease (Bluestone, 2005).T_(Reg) cells express CD4 and CD25 as wells as the forkheadtranscription factor boxP3 (Foxp3) (Sakaguchi, 2005), which serves as amaster regulator for T_(Reg) development and function (Fontenot et al.,2003; Hori et al., 2003). Indeed, Foxp3-mutant mice have a deficiency inT_(Reg) cells and develop severe lymphoproliferative autoimmunesyndrome. Similarly, humans with the rare recessive disorder:Immunodysregulation, Polyendocrinopathy and Enteropathy X-linked (IPEX)syndrome exhibit aggressive autoimmunity and early (Walker et al.,2003).

T_(Reg) cells can be generated both in the thymus and in the peripheryand appear phenotypically and functionally similar. Studies withTCR-transgenic systems indicate that relatively high-affinityinteractions of αβTCR with self-peptide agonists presented on thymicepithelial cells are required to efficiently generate T_(Reg) cells in aCD28-dependent manner (Apostolou et al., 2002; Jordan et al., 2001; Taiet al., 2005; Walker et al., 2003). As a result, intrathymic T_(Reg)cells utilize a diverse TCR repertoire (Bluestone and Abbas, 2003)skewed toward autoantigen recognition. Recently, it was demonstratedHassall's corpuscles express thymic stromal lymphopoietin (TSLP), whichactivates thymic dendritic cells to induce the proliferation of T_(Reg)cells (Watanabe et al., 2005). Alternatively, T_(Reg) cells can beexpanded extrathymically through differences in self-peptide exposureand cytokine milieu (i.e.: transforming growth factor-β (TGF-β) andIL-10) (Apostolou and von Boehmer, 2004; Belghith et al., 2003;Roncarolo et al., 2001; Weiner, 2001).

The observation that T_(Reg) cells are deficient in patients withmultiple sclerosis, type 1 diabetes, rheumatoid arthritis (Ehrenstein etal., 2004; Lindley et al., 2005; Viglietta et al., 2004) has raised hopethat treatment of these and other autoimmune diseases may rest with therestoration of T_(Reg) cells (Bluestone, 2005). In contrast, theelimination of T_(Reg) cells may play a significant role in enhancingcancer immunotherapeutic approaches by releasing the breaks on antitumorT cell responses and inducing limited local autoimmunity (Sakaguchi etal., 2001). Finally, T_(Reg) cells may play a critical role in theestablishment of tolerance following allogenic organ transplant therebyminimizing rejection mediated by GvHD (Gregori et al., 2005; Hoffmannand Edinger, 2006; Touraine et al., 2005).

As with most cellular based therapies, the major obstacle for theutilization of T_(Reg) cells in the treatment of autoimmunity is theability to generate them in large numbers to realize therapeuticeffectiveness. Currently, the OP9-DL1 coculture system does not supportthe generation of large numbers of T_(Reg) cells from progenitor Tcells. Given the role of TSLP in the generation of T_(Reg) cells(Watanabe et al., 2005), it is unclear whether the absence of T_(Reg)cells in the OP9-DL1 coculture system is due to a deficiency of OP9cells to produce TSLP.

Regardless, stem cell transplantation for the treatment of severeautoimmunity is gaining momentum (Bluestone, 2005; Gregori et al., 2005;Sykes and Nikolic, 2005) with the development of human/immunodeficientmouse models of alloreaction (Thomsen et al., 2005), methods to expandregulatory T cell populations (Kretschmer et al., 2005) and to engineerstem cells and progenitor T cells to express self-antigen (Alderuccio etal., 2003).

Accordingly, the present application provides a method of treating orpreventing an autoimmune disease comprising administering an effectiveamount of a progenitor T cell to an animal thereof.

(iv) Genetic Diseases

As mentioned previously, the pro-T cells may be transfected with adesired gene. Such cells can be used for treatment of genetic diseases.Hematopoietic cell-related genetic diseases can be treated by graftingthe cellular composition with cells transfected with a gene that canmake up for the deficiency or the abnormality of the gene causing thediseases. For example, a normal wild type gene that causes a diseasesuch as β-thalassemia (Mediterranean anemia), sickle cell anemia, ADAdeficiency, recombinase deficiency, recombinase regulatory genedeficiency and the like, can be transferred into the pro-T cells byhomologous or random recombination and the cells can be grafted into apatient. Further, a cellular composition comprising normal T cells freefrom abnormalities of genes (from a suitable donor) can be used fortreatment.

Another application of gene therapy permits the use of a drug in a highconcentration, which is normally considered to be dangerous, byproviding drug resistance to normal T cells by transferring a drugresistant gene into the cells. In particular, it is possible to carryout the treatment using an anticancer drug in high concentration bytransferring a gene having drug resistance against the anticancer drug,e.g., a multiple drug resistant gene, into pro-T cells in a cellularcomposition of the application.

Diseases other than those relating to the hematopoietic system can betreated by using the cellular compositions comprising pro-T cells in sofar as the diseases relate to a deficiency of secretory proteins such ashormones, enzymes, cytokines, growth factors and the like. A deficientprotein can be induced and expressed by transferring a gene encoding atarget protein into the pro-T cells under the control of a suitablepromoter. The expression of the protein can be controlled to obtain thesame activity as that obtained by the natural expression in vivo.

It is also possible to insert a gene encoding a ribozyme, an antisensenucleic acid or the like (e.g., short-interfering RNA) or anothersuitable gene into pro-T cells to control expression of a specific geneproduct in the cells or to inhibit susceptibility to diseases. Forexample, the pro-T cells can be subjected to gene modification toexpress an antisense nucleic acid, siRNA, or a ribozyme, which canprevent growth of hematic pathogens such as HIV, HTLV-I, HTLV-II and thelike in pro-T cells. In an embodiment, pro-T cells of a cellularcomposition of the application are created which express knowninhibitory genes of HIV replication, such as RNA decoys or the Tat- orRev-responsive elements, or a dominant negative mutant of the Revtrans-activator protein. Pro-T cells derived from hematopoieticprogenitor cells or ES carrying these genes would provide a potentiallylimitless and defined source of HIV-resistant lymphocyte progenitors.

C. Screening

The cellular compositions comprising pro-T cells may be used to screenfor potential modulators or therapeutics that modulate development oractivity of pro-T cells or cells differentiated therefrom. Inparticular, the cellular compositions may be subjected to a testsubstance, and the effect of the test substance may be compared to acontrol (e.g. in the absence of the substance) to determine if the testsubstance modulates development or activity of pro-T cells or cellsdifferentiated therefrom.

In an aspect of the application a method is provided for using acellular composition of the application comprising pro-T cells or cellsdifferentiated therefrom to assay the activity of a test substancecomprising the steps of:

-   -   (a) generating pro-T cells with a system or method of the        application in the presence of a test substance, or culturing        pro-T cells compositions generated using a system or method of        the application in the presence of a test substance; and    -   (b) detecting the presence or absence of an effect of the test        substance on the survival of the cells or on a morphological,        functional, or physiological characteristic and/or molecular        biological property of said cells, whereby an effect altering        cell survival, a morphological, functional, or physiological        characteristic and/or a molecular biological property of the        cells indicates the activity of the test substance.

In another aspect a method is provided for using pro-T cells or cellsdifferentiated therefrom generated in accordance with the application,to screen a potential new drug to treat a disorder involving T cellscomprising the steps of:

-   -   (a) generating pro-T cells with a system or method of the        application in the presence of a potential new drug, or        culturing pro-T cells preparations generated using a system or        method of the application in the presence of a potential new        drug; and    -   (b) detecting the presence or absence of an effect of the        potential new drug on the survival of the cells in vitro or on a        morphological, functional or physiological characteristic and/or        molecular biological property of said cells, whereby an effect        altering cell survival, a morphological, functional, or        physiological characteristic and/or a molecular biological        property of the cells in vitro indicates the activity of the        potential new drug.

The cellular compositions of the application may be used to preparemodel systems of disease. The cellular compositions of the applicationcan also be used to produce growth factors, hormones, etc.

The cellular compositions of the application can be used to screen forgenes expressed in or essential for differentiation of T cells.Screening methods that can be used include Representational DifferenceAnalysis (RDA) or gene trapping with for example SA-lacZ (D. P. Hill andW. Wurst, Methods in Enzymology, 225: 664, 1993). Gene trapping can beused to induce dominant mutations (e.g. by deleting particular domainsof the gene product) that affect differentiation or activity of T cellsand allow the identification of genes expressed in or essential fordifferentiation of these cells.

The following non-limiting examples are illustrative of the presentapplication:

EXAMPLES Example 1 Preparation of Umbilical Cord Blood Sample

Obtain 25-50 mls of human umbilical cord blood (UCB) by syringeextraction and collect in a single blood pack unit containing citratephosphate dextrose anti-coagulant (CPDA) (Baxter Healthcare, Deerfield,Ill.). Within 12 hours of collection, cord blood mononuclear cells areisolated using Ficoll density centrifugation and frozen until furtheruse. Specifically, the human cord blood sample is diluted 1:4 in PBS orHBSS+2 mM EDTA. The mononuclear cells are isolated by gradientcentrifugation in Ficoll-Paque Plus (Amersham Biosciences, Cat17-1440-03). Using a thin sterile Pasteur Pipette underlay the dilutedcord blood sample with Ficoll-Paque. Centrifuge at 1350-1860 rpm for30-40 minutes at 18-20° C. Wash lymphocyte layer 3× in PBS or HBSS,centrifuging between each wash at 1200 rpm for 5 minutes, removingsupernatants each time. Resuspend cells in 1 ml of sterile FACS sortingbuffer and freeze at −80° C. For each experiment, frozen UCB was thawedand then pre-enriched into lineage-negative (Lin−) and lineage-positive(Lin⁺) fractions with the autoMACS™ or autoMACS-pro separator (MiltenyiBiotec, Auburn, Calif.) using the StemSep® human progenitor cellenrichment cocktail (Stem Cell technologies, Vancouver, BC, Canada). Toisolate the human HSCs, Lin⁻ cells were stained with anti-human CD38-APCmAbs and anti-human CD34-PE mAbs and subsequently sorted forCD34⁺CD38^(−/lo) cells utilizing a BD Biosciences FACSAria digital cellssorter (San Jose, Calif.). Sorted human HSCs were greater than 99% pureas determined by post-sort analysis.

Example 2 Human Hematopoietic Stem Cells and OP9-DL1 or OP9-DL4 CellCoculture

OP9 cells retrovirally-transduced to express either GFP-vector backbone(OP9-control) or bicistronic plasmid containing GFP and Delta-like 1(OP9-DL1) or Delta-like 4 (OP9-DL4) were generated as previouslydescribed (Schmitt and Zúñiga-Pflücker, 2002), and maintained in α-MEMmedium supplemented with 20% coculture-characterized fetal bovine srum(FBS) (Hyclone), plus 50 U/ml penicillin and 50 μg/ml streptomycin(OP9-media). In most experiments, 1-5×10⁴ sorted human HSCs(CD34⁺CD38^(−/lo)) were added per individual well of a 6-well platecontaining confluent OP9-DL1 or OP9-DL4 cells, and cultured in OP9-mediasupplemented with recombinant human cytokines Flt-3L (5 ng/ml) and IL-7(5 ng/ml), (Peprotech, Rocky Hill, N.J.). Every 4-5 days, humanHSCs/(OP9-DL1 or OP9-DL4)cocultures were transferred onto a freshconfluent monolayer of OP9-DL1 or OP9-DL4 cells. For high cell densitycocultures, media changes were performed every 2 days between passagesas previously described (Awong et al., 2008).

The ability to utilize simple stromal cell monolayers that expressDelta-like-molecules such as OP9-DL1 cells or S17-DL1 cells haspermitted a closer examination of human T cell development than waspreviously possible. The OP9-DL1 cells have been already distributed tonearly 400 laboratories around the world. Research emerging from theselaboratories has confirmed the molecular elements required to sustainthe generation of T cells (Lehar and Bevan, 2002; Wang and Spangrude,2003) from numerous sources of progenitor cells and has helped toelucidate many other factors with critical roles in T cell development(Gutiérrez-Frias et al., 2004; Outram et al., 2000; Pongracz et al.,2003; Shah et al., 2004; Staal et al., 2004; Weerkamp et al., 2006b;Weerkamp et al., 2006d).

While the paradigm that T cell development requires signaling throughDelta-like ligands has been firmly established for Delta-like-1expression, it is beginning to emerge that Delta-like-4 expression canalso recapitulate T cell development in vitro (Schmitt andZúñiga-Pflücker, 2006). This is not surprising given that Delta-like-4shares sequence homology with Delta-like-1 and is also expressed withinthe thymus (Schmitt and Zúñiga-Pflücker, 2002).

Prior to the advent of OP9-DL1 or OP9-DL4 cells, the study of human Tcell development required cumbersome FTOCs and their derivative systems,which although functional, were cumbersome, inefficient, and impracticalgiven the limitations of hybrid/mouse FTOCs and the lack of availablehuman fetal thymic tissue. Thus, it was difficult to conceive how onecould generate the numbers of human progenitor T cell needed to elicittherapeutic effectiveness for the treatment of immune-related disease.The OP9-DL1 and OP9-DL4 technology has numerous advantages. Many ofthese advantages have been reviewed previously with an emphasis on mouseT cell development (Zúñiga-Pflücker, 2004) and offer similar advantageswith regard to the study of human T cell development.

An important practical consideration of the OP9-DL1 and OP9-DL4coculture systems is its technical simplicity. Human HSCs are cultureddirectly on a simple monolayer with the addition of two human cytokinesFlt-3L and IL-7 for long periods of time. Thus, with media changes andtransfer onto new stroma, the OP9-DL1 and OP9-DL4 cells are easilymanipulated and readily expanded into larger cultures. This is incontrast to cumbersome FTOCs, which often require direct microinjectionof human progenitor cells and have limited culture times. Given theseconstraints, the OP9-DL1 and OP9-DL4 coculture system represents animprovement over FTOCs in that a single cell can be now assayed for Tcell progenitor cell function (Ciofani et al., 2006; Schmitt andZúñiga-Pflücker, 2002). Although this was possible using FTOCs (Ikawa etal., 1999; Michie and Zúñiga-Pflücker, 2000; Williams et al., 1986), thefeasibility of large-scale analyses was nearly prohibitive and therewere no reports of progenitor frequency using single cell analysis forhuman progenitor T cells. Thus, together with the current assayspresently used in FTOCs, the inventors' system can complement differentapproaches utilized in the study of human T cell development, openingnew avenues for future research to test the immune reconstitutioncapabilities and immune function of in vitro-derived T cells (Jenkinsonand Anderson, 1994; Takahama, 2000).

Additionally, the OP9-DL1 and OP9-DL4 systems have been able to supportgeneration of T cells from a number of defined sources. Mouse progenitorcells obtained from fetal liver, bone marrow, fetal thymus, andperipheral blood, and embryonic stem cells (ESCs) generate T cells uponOP9-DL1 coculture (Adolfsson et al., 2005; Ciofani and Zúñiga-Pflücker,2005; Schmitt et al., 2004; Schmitt and Zúñiga-Pflücker, 2002).Similarly, human progenitor cells isolated from fetal liver, bonemarrow, fetal thymus, GM-CSF-mobilized peripheral blood, and umbilicalcord blood generate T cells upon OP9-DL1 coculture (De Smedt et al.,2004; La Motte-Mohs et al., 2005; Weerkamp et al., 2006a). With regardto ESCs and HSCs, the emerging uses of short interfering RNA (siRNA)(Gimeno et al., 2004; McManus and Sharp, 2002) and locked nucleic acids(Grünweller et al., 2003) as methods of choice to quickly assay for thefunctional importance of a particular gene, for instance during T celldevelopment, can be easily adapted to the OP9-DL1 and OP9-DL4 cellcoculture systems. This allows for a practical approach to characterizethe role during T cell development of many genes that when deleted,result in an embryonic lethal phenotype and simply cannot be furtherstudied. Similarly, the OP9-DL1 system is adaptable to geneticengineering. This principle has been demonstrated with retroviral andlentiviral vectors in CD34⁺ HSC (immature) (Case et al., 1999; Gimeno etal., 2004; Haas et al., 2000; Klug et al., 2000; Su et al., 1997), butnot progenitor T cells. As progenitor T cells are cycling and renewed inthe OP9-DL1 coculture system, it is likely they will prove equallyamenable to genetic manipulation. Thus, together with the current assayspresently used with mouse T cell development, there are many differentapproaches that can now be easily applied to the study of human T celldevelopment.

In contrast to mouse ESCs, undifferentiated human ESCs have not yetgenerated progenitor cells that can give rise to T cells followingcoculture with either OP9-DL1 or OP9-DL4 cells. Indeed, thedifferentiation of human ESCs in OP9-DL1 coculture has proven morechallenging. Several groups have demonstrated efficient differentiationof human ESCs into CD34⁺ cells through embryonic body formation (Cerdanet al., 2004; Chadwick et al., 2003; Wang et al., 2004; Zambidis et al.,2005; Zhan et al., 2004) or coculture on S17 (Tian et al., 2006), MS5 orOP9 (Vodyanik et al., 2005) stromal lines. These CD34⁺ cells when sortedand re-cultured onto bone marrow stroma give rise to B (Vodyanik et al.,2005) and NK {Woll, 2005 #219(Vodyanik et al., 2005)} cells anddendritic cells (Slukvin et al., 2006) in the presence of theappropriate cytokine, suggesting that human ESCs can differentiate intoprogenitor cells with multilineage potential. In contrast, sorted humanESC-derived CD34⁺ cells have thus far not differentiated in vitro into Tcells upon coculture with OP9-DL1 cells, nor have embryonic body-derivedCD45⁻, PECAM-1⁺, Flk-1⁺, and VE-cadherin⁺ (PFV) cells upon intra-femoralinjection into immunodeficient NOD/SCID mice (Wang et al., 2005b),suggesting that human ESCs may be less sensitive in vitro toDelta-like-Notch induced differentiation signals than mouse ESCs topromote T cell differentiation. Alternatively, the OP9-DL1 cells may notcompletely substitute for all of the factors required for the inductionand early differentiation of human ESCs. This issue has recently beencircumvented in vivo through the direct injection of human ESC-derivedGFP-labeled CD34⁺ cells into conjoint human thymic/liver (Thy/Liv)tissues implanted under the kidney capsule into sublethally irradiatedimmunodeficient SCID-mice (Fleming and Scadden, 2006; Galic et al.,2006).

The ability to derive T cells from human ESCs remains an attractive goalfor the treatment of immune-related disorders. This is due to thegeneral consensus that theoretically, unlimited numbers of human T cellsmay be generated from undifferentiated human ESC cell lines, whereashuman HSCs may possess only limited differentiation and proliferationpotential from exhaustible sources such as bone marrow and cord blood.Foresight not withstanding, only a small number of human HSCs can beisolated from limited tissue and therefore must be properly stored andexpanded for future use. Efforts to expand human HSCs numbers whilepreventing their differentiation are under development and could beutilized in concert to generate even greater numbers of progenitor Tcells. Taken into consideration that not all HSCs exhibit long-termreconstitution potential, and the thymus does not contain progenitorcells that self-renew, the utilization of HSCs and progenitor T cellsmay have limited niches for the treatment of T cell basedimmunodeficiency or autoimmunity. Human ESCs are not without theirdrawbacks and serious concerns remain about their safety given theirgenomic instability, epigenetic status, their propensity spontaneousdifferentiation and their potential to cause cancer (Odorico et al.,2001; Olsen et al., 2006; Rugg-Gunn et al., 2005; Wang et al., 2005a).

In the OP9-DL1 or OP9-DL4 coculture systems, human HSCs derived fromcord blood and bone marrow show robust expansion. Indeed, the OP9-DL1 orthe OP9-DL4 system generates a population of T lineage cells that arehighly homogenous and easily isolated based on human markers of T celldifferentiation. This system generates T lineage cells efficiently(>90%) at the expense of other lymphocytes and myeloid cells; however,the upper-limit of this T cell expansion is unknown. The output ofprogenitor-T cells in this coculture system is at least 10³-10⁵ timeshigher than other in vitro systems suggesting that further scaling-up ofthis system could yield clinically relevant numbers to achievetherapeutic benefits in patients with immune related disorders. Longterm OP9-DL1 cocultures initiated with human CD34⁺CD38⁻ cellsdemonstrate sustained T cell development for at least 120 days andretain a population of cells, which are CD34⁺CD7⁺. Whether these cellsare capable of self-renewal is unclear, although multiple waves of Tcell development have been observed in long-term OP9-DL1 or OP9-DL4cocultures [La Motte-Mohs, unpublished observations]. These waves of Tcell development could simply reflect apoptosis of CD4⁺CD8⁺ DP T cellpopulations that did not receive positive and negative selection signalsfollowed by the re-emergence of progenitor T cells derived from earlyprogenitors maintained in the cultures. Indeed, such a possibility isconsistent with the dual role of Notch signaling in maintainingprogenitor cell renewal and promoting T cell differentiation(Varnum-Finney et al., 1998). Further studies are required to determinewhether the OP9-DL1 or OP9-DL1 coculture systems promote both T celldifferentiation and self-renewal at the same time

Although the differentiation from stem cell to functional T cell can beobtained readily by coculture on OP9-DL1 cells (Schmitt et al., 2004;Schmitt and Zúñiga-Pflücker, 2002), there are still some drawbacks tothis system. For instance, OP9 cells express mouse MHC class I moleculesand support the differentiation of mouse HSCs into CD8⁺ T cells, but donot express mouse MHC class II molecules and do not appear to expressCD1d; thus, limiting their ability to support the differentiation of CD4T cells and NKT cells, respectively (Schmitt and Zúñiga-Pflücker, 2002;Zúñiga-Pflücker, 2004). While there is evidence that mouse MHC moleculescan support the differentiation of human HSCs (Fisher et al., 1990;Traggiai et al., 2004), this could be especially problematic whendeveloping cellular immune-therapies to treat immune-related disorderswithout invoking an autoimmune response or graft-verses host disease(GvHD). However, OP9-DL1 cells could be modified to ectopically expresshuman MHC molecules, which would permit a re-examination of thecontributions of these molecules to the development of specific subsetsof T cells as well as generate MHC-matched T cells to an individual.Specifically, the inventors have reported the robust and sustainedgeneration of human T cells to the DP stage from cord blood-derivedHSCs; however, the generation of CD4⁺ or CD8⁺ SP T cells has beenlimited (La Motte-Mohs et al., 2005), which is likely due to the absenceof human HLA molecules on mouse OP9-DL1 cells. Strikingly, underlong-term densely packed coculture conditions the inventors can detectCD4⁺ or CD8⁺ T cells that express TCRαβ unpublished observations, RossLa Motte-Mohs]. At first glance, the emergence of human single positiveT cells seems difficult to reconcile given the published reports thatisolation of human progenitor T cells require human thymic stromalelements to realize their full differentiation potential towards SP Tcells. Nevertheless, a recent paper by Choi et al demonstrated thatthymocyte-thymocyte (T-T) interactions can mediate positive selectionand promote the maturation of CD4 T cells in the absence of MHC-classII⁺ thymic stroma (Choi et al., 2005). Similarly, the emergence of humanCD4⁺ SP T cells during high-density coculture conditions with OP9-DL1cells may take advantage of T-T interactions as developing progenitor Tcells (CD34⁺CD7⁺) express high levels of human MHC class II molecules[unpublished observations, Ross La Motte-Mohs. Alternatively, γδ-Tcells, which emerge later in OP9-DL1 coculture may function asprofessional APCs towards emerging αβ-T cells permitting furtherdifferentiation (Brandes et al., 2005; Modlin and Sieling, 2005).

Interestingly, another drawback is the limited number of self-antigensthat OP9-DL1 cells are likely to present to developing T cells.Initially, OP9 cells, in contrast to thymic medullary epithelial cells,were thought unlikely to express the AIRE gene (Anderson et al., 2002)and mediate ectopic self-antigen presentation for peripheral tolerance.However the detection of low levels AIRE message in OP9-DL1 cells[personal communication, Lynn Rumfelt] suggests that OP9-DL1 cells mayposses some capacity to present tissue-specific-antigens, such asinsulin, which was also detected in OP9 cells. Whether the AIREtranscription factor is functional in OP9-DL1 cells remains to beconfirmed experimentally. However, the recent demonstration that skincells that express AIRE and Delta-like-1 can support thymic-independentT cell development and mediate negative selection (Clark et al., 2005),suggests a similar possibility for the OP9-DL1 cells. Thus, questionsdealing with the mechanisms responsible for positive and/or negativeselection of the TCR repertoire can be investigated by appropriatelymanipulating the OP9-DL1 cells and are currently under investigation inthe inventors' laboratory. Nonetheless, issues related to the ability orfunction of OP9-DL1 cells to properly select mature T cells can beavoided by simply transferring CD4⁻ CDT double negative progenitors orimmature CD4⁺CD8⁺ T cells, obtained from stem cells cultured on OP9-DL1cells, into FTOC or intrathymically into host mice (Schmitt et al.,2004). Such an approach not only provides a practical solution to theself-MHC restriction and tolerance issues, but also opens new avenuesfor future possibilities to test the immune function of the invitro-derived T cells as well as their efficacy in adapting T cells totreat human immune-related disorders.

These studies underline valid concerns to determine the efficacy andtherapeutic effectiveness for the utilization of in vitro-derivedprogenitor T cells for the treatment of immune-related disorders.Progenitor T cells, whether autologous, or allogeneic, generated in theOP9-DL1 system are immature and still need to undergo positive andnegative selection in the host thymus. This suggests that they areunlikely to evoke an auto-immune response or GvHD in vivo. Although GvHDremains a concern in stem cell transplantation, the reconstitution of ahuman immune system using CD34⁺ HSC, but not progenitor T cells, in bothpatients and immunodeficient mice have demonstrated the principle ofthis approach (Barker and Wagner, 2003; de Wynter et al., 1999; Gimenoet al., 2004; Traggiai et al., 2004). Indeed, the utilization ofhuman/mouse models of engraftment (Legrand et al., 2006) may proveparticularly useful in helping to evaluate the safety of in vitroderived human progenitor T cells for the treatment of immune-relateddisorders.

Example 3 Human-Mouse Fetal Thymic Organ Coculture (FTOC)

FTOC (Fisher et al., 1990; Plum et al., 2000) was performed by isolatingfetal thymuses from embryos of time-pregnant CD1 mice at day 15 ofgestation (Jackson Laboratories, Bar Harbor Me.). The thymuses werecultured for 5 days in the presence of 1.35 mM deoxyguanosine (dGuo) toremove endogenous thymocytes. Human pro-T subsets derived fromHSC/OP9DL1 cocultures supplemented with Flt-3L (5 ng/ml), IL-7 (5 ng/ml)and SCF (30 ng/ml) (Peprotech, Rocky Hill, N.J.) were sorted and placedinto hanging drops in Terasaki wells for 24 hours followed by transferto Nucleopore filters on Gelfoam rafts for 7-21 days as indicated.OP9-media and cytokines (Flt-3L and IL-7) were replenished every 5 days.Cells were analyzed by crushing the thymic lobes with a nylon mesh cellstrainer to obtain single-cell suspensions.

Traditionally, the development of in vitro models to study human Tlymphopoiesis and progenitor cell commitment have relied on the use ofhost thymic tissue obtained from fetal mice, or from electivelyterminated human fetuses, and from thymus tissue discarded duringpediatric cardiac surgeries. Until recently, the only in vitro modelsystem that permitted the generation of human T cells was the hybridFTOC system first adapted by Fisher et al (Fisher et al., 1990). In thiswhole organ based approach, embryonic day 14/15 mouse thymic lobes aredepleted of endogenous thymocytes through treatment with2-deoxyguanosine, seeded with hematopoietic progenitor cells via thehanging drop method and cultured for a period of time on GelFoam-rafts.T cell developmental stages can then be assessed at various time pointsfollowing introduction of murine hematopoietic progenitors into thethymic rudiments. Using this hybrid human/mouse FTOC, Fisher et aldemonstrated the proliferation and generation of mature SP T cells fromhuman fetal thymic progenitor cells (Fisher et al., 1990). This approachwas also demonstrated later with postnatal human progenitor thymocytes(Merkenschlager and Fisher, 1991) and human progenitor cells obtainedfrom bone marrow and cord blood (Yeoman et al., 1993). The study byFisher et al noted that efficient colonization of murine thymicrudiments by human thymic progenitor cells depended on the addition ofhuman thymic stromal elements (Fisher et al., 1990). In order to improvethymic colonization and T cell development, several groups directlyinjected human progenitor cells into human fetal thymic fragments (Galyet al., 1993; Peault et al., 1991). Despite its inefficiency andtechnical complexity, hybrid human/mouse FTOCs are routinely used toexamine human hematopoiesis and T cell differentiation (Barcena et al.,1995; De Smedt et al., 2002; Galy et al., 1993; Plum et al., 1994; Reset al., 1997).

Example 4 Quantitative Real-Time Reverse-Transcriptase Polymerase ChainReaction (Q-PCR)

Total RNA was isolated in Trizol reagent and reverse transcribed usingSuperscript III and Oligo(dT)₁₂₋₁₈ primers (Invitrogen, Burlington, ON).Diluted cDNA samples from total OP9-control cocultures, total OP9-DL1cocultures, sorted T-lineage subsets from OP9-DL1 cocultures asindicated in the figures, UCB purified Lin⁺CD3⁺ and CD33⁺ cells, or bulkand Lin⁻ human post-natal thymocytes (PNT) were used as templates forQ-PCR reactions. Detection of the Q-PCR was performed with the SYBRGreen PCR master mix according to manufacturer's instructions (Qiagen,Mississauga, Ontario or Bio-Rad, Hercules Calif.) on the AppliedBiosystems Sequence Detection System 7000. All transcript levels werenormalized to human β-actin. Gene-specific forward (F) and reverse (R)primers are as follows:

Rag-1, (F) (SEQ ID NO: 1) CAACCAAATTGCAGACATCTCAAC and (R)(SEQ ID NO: 2) CCATGCTGGCTGAGGTACCT; Deltex-1 (F) (SEQ ID NO: 3)GTGAGCAAGAGCGACGTGAAG and (R) (SEQ ID NO: 4) ACCACATCCTCGGGATTCTTACT;Notch-1 (F) (SEQ ID NO: 5) CGGGTCCACCAGTTTGAATG and (R) (SEQ ID NO: 6)GTTGTATTGGTTCGGCACCAT; Gata-3 (F) (SEQ ID NO: 7) GATGGCACGGGACACTACCTand (R) (SEQ ID NO: 8) GCTCTCCTGGCTGCAGACA; Cebpα (F) (SEQ ID NO: 9)CGGACTTGGTGCGTCTAAG  and (R) (SEQ ID NO: 10) GAGGCAGGAAACCTCCAAAT; Ccr9,(F) (SEQ ID NO: 11) TGTCCCAGGGAGAGTTGCA and (R) (SEQ ID NO: 12)GGGTGTCATGGTGGGTCAGT; Selplg (F) (SEQ ID NO: 13) GTGCCATGCCTCTGCAACT and(R) (SEQ ID NO: 14) TGTCCCACAGCTGCAAGCT; Itga2 (F) (SEQ ID NO: 15)TCTGAGACTGCCAAGGTCTTCA and (R) (SEQ ID NO: 16) CAGCTGGTATTTGTCGGACATC;Itga4 (F) (SEQ ID NO: 17) AAGCTGACTGTTCATGGGTTTGT and (R)(SEQ ID NO: 18) TCTCCACCATGCACGTTTCA; Itga5 (F) (SEQ ID NO: 19)CAGTGCCGAGTTCACCAAGA and (R) (SEQ ID NO: 20) GCCTTGCCAGAAATAGCTTCCT;Itgb1 (F) (SEQ ID NO: 21) TCAGAATTGGATTTGGCTCATTT and (R)(SEQ ID NO: 22) CCTGAGCTTAGCTGGTGTTGTG; and  β-actin (F) (SEQ ID NO: 23)TTGCCGACAGGATGCAGAA and (R) (SEQ ID NO: 24) GCCGATCCACACGGAGTACT.

Example 5 Flow Cytometry

Fluorescein isothiocyanate (FITC)-, R-Phycoerythrin (PE)-,allophycocyanin (APC)-, PE-Cy7-, Peridinin chlorophyllprotein (PerCP)PerCP-Cy5.5-, Alexa Fluor₇₀₀-, Alexa Fluor₇₅₀-, and PacificBlue-conjugated antibodies were purchased commercially. They include thefollowing antibodies: FITC: anti-CD34 (clone 581), anti-CD27 (cloneM-T271), anti-CD3 (clone HIT3a), anti-TCRαβ (clone T10B9.1A-31); PE:anti-CD7 (M-T701), anti-CD4 (clone RPA-T4), anti-CD49d (clone 9F10)anti-granzyme B (clone eBioGrB); APC: anti-CD1a (clone HI149), anti-CD7(CD7-6B7), anti-CD8 (clone RPA-T8); PE-Cy7: anti-CD8 (clone RPA-T8);PerCP-Cy5.5: anti-CD5 (clone L17F12); Alexa Fluor₇₀₀: anti-CD4 (cloneRPA-T4); APC-Cy7/APC-Alexa Fluor₇₅₀: anti-CD4 (clone RPA-T4); PacificBlue: anti-CD3 (clone UCHT1). Intracellular staining for granzyme B wasperformed using the Cytofix/Cytoperm kit according to manufacturer'sinstructions (BD Biosciences, San Diego, Calif.). All antibodies wereobtained from BD Pharmigen with the exceptions of anti-CD49d-PE,anti-granzyme B-PE, anti-CD3-FITC and anti-CD4-APC-Alexa Fluor₇₅₀, whichwere purchased from eBioscience (San Diego, Calif.). For flow cytometricanalyses, cell suspensions obtained from OP9-DL1 cocultures, or fetalthymic organ cultures (FTOCs) were FcR11 blocked and stained. Cells wererun on a FACSCalibur (BD-Biosciences) or a four-laser LSR II benchtopflow cytometer. Data analysis was performed using FlowJo software (TreeStar, Ashland, Oreg.) by gating on live lymphocytes and lack ofpropidium iodide uptake. GFP-expressing OP9 stromal cells were excludedthrough GFP expression and side scatter gating. This procedureeliminated 99% of contaminating GFP-expressing OP9 stromal cells.Numbers in quadrant corners represent percent of gated cells.

Example 6 T Cell Stimulation Assays

In vitro-generated CD3/TCR-αβ⁺CD8⁺ single positive (SP) cells weresorted from HSC/OP9-DL1 cocultures at days 60-70. For T cell stimulationassays, 4×10⁴ cells were seeded in individual wells of a flat bottom96-well plate coated with or without anti-CD3 (2 or 10 μg/ml) andsoluble anti-CD28 (1 μg/ml) mAbs. All wells contained OP9-mediasupplemented with recombinant human IL-2 (1 ng/ml) and recombinant humanIL-7 (1 ng/ml) cytokines and were analyzed after 5 days. For T cellproliferation assays, 4×10⁴ in vitro-generated CD8⁺ T cells were sortedand loaded with 10 μM carboxyfluorescein succinimidyl ester (CFSE)according to manufacturer's protocol (Molecular Probes, Eugene, Oreg.)prior to plating. Loss of CFSE labeling was assayed after 5 days ofstimulation using a FACSCalibur flow cytometer.

Example 7 Precursor Frequency Analysis

Human HSC limiting dilution assay (LDA) was performed by serialdilutions from different cell subsets of UCB samples. UCB cells weresorted as CD34⁺CD38⁻, CD34⁺CD38^(lo), CD34⁺CD38^(+/hi) using theFACSDiVa cell sorter, and 1 (n=36), 3 (n=24), 10 (n=90), 30 (n=56), 100(n=58) or 300 (n=13) cells of each subset were directly deposited intoindividual wells of a 96 well/plate containing OP9-DL1 cell monolayers.Cells were cultured for 11 days, after which they were harvested fromindividual wells and analyzed by flow cytometry. The presence ofCD45⁺CD7⁺⁺ cells was scored, and the progenitor frequency determined bythe method of maximum likelihood applied to the Poisson model (Fazekasde St, 1982). For human in vitro-derived progenitor-T cells limitingdilution assays were performed using sorted CD34⁺ CD7⁺⁺CD5⁻ andCD34⁺CD7⁺⁺CD5⁺ subsets obtained from a day 13 HSC/OP9-DL1 coculture, andseeded into a dGuo-FTOC-derived thymus lobe at 500 (n=2), 1000 (n=18),1500 (n=12), 2000 (n=13), 3000 (n=13), 9000 (n=4) or 22000 (n=1) cellsper lobe for CD34⁺CD7⁺⁺CD5⁻ progenitors or 100 (n=4), 300 (n=9), 500(n=10), 1000 (n=10), 3000 (n=10), 9000 (n=4) or 22000 (n=1) cells perlobe for CD34⁺CD7⁺⁺CD5⁺ progenitors. Progenitors were also seeded backonto OP9-DL1 cells in 96 well/plates, and deposited at 1 (n=36), 3(n=20), 10 (n=20), 30 (n=14), and 100 (n=6) cells per well. Cells wereanalyzed after 7 days of differentiation, and scored for the presence ofCD45⁺CD7⁺⁺ (FTOC) or CD7⁺⁺CD1a^(−/+) cells (OP9-DL1) cells. Theprogenitor frequency was determined by the method of maximum likelihoodapplied to the Poisson model (Fazekas de St, 1982).

Example 8 Thymic Reconstitution of Immunodeficient Mice by HumanProgenitor Cells Generated In Vitro Materials & Methods

Umbilical Cord Blood Samples:

Human UCB samples were obtained by syringe extraction and collected in ablood-pack unit containing citrate phosphate dextrose anti-coagulant(Baxter Healthcare, Deerfield, Ill.) from consenting mothers followingdelivery at Women's College Hospital in accordance to approvedguidelines established by the Research Ethics Board of Sunnybrook HealthSciences Centre. Within 12 hours of collection, UCB mononuclear cellswere isolated by Ficoll density centrifugation. For each experiment,frozen UCB was thawed and pre-enriched into lineage-negative (Lin−) andlineage-positive (Lin⁺) fractions with the autoMACS™ (Miltenyi Biotec,Auburn, Calif.) using the StemSep® enrichment cocktail (Stem Celltechnologies, Vancouver, BC, Canada). To isolate human HSCs, Lin⁻ cellswere stained with anti-human CD38-APC and anti-human CD34-PE mAbs andsubsequently sorted for CD34⁺CD38^(−/lo) cells using a BD BiosciencesFACSAria sorter (San Jose, Calif.). Sorted human HSCs were greater than99% pure as determined by post-sort analysis.

NOD/SCIDγc^(−/−) and RAG2^(−/−)γc^(−/−) Reconstitution Studies:

5-6×10⁵ sorted HSCs)(CD34⁺CD38^(−/lo) were added at 3×10⁴ cells perindividual well of a 6-well plate containing confluent OP9-DL1 cells,and the cultures were maintained for 10-12 days in the presence of OP9media plus rhlL-7 (5 ng/mL); rhFlt-3L (5 ng/mL) and rhSCF (30 ng/mL)after which CD34⁺CD7⁺ progenitor T cells (ProT) were sorted. Sortedhuman pro-T cells were resuspended in recombinant human IL-7/M25 mixture(provided by Dr. C. Surh) and 3.5-5×10⁵ cells injected (30 μl/mouse)intrahepatically into 4-5 day old neonates. As controls, mice wereinjected with either PBS or CD34⁺ stem cells (1.5-2.5×10⁵). Mice wereboosted with IL-7/M25 mixture every 3-4 days. Thymus, spleen and bonemarrow were harvested 21-27 days after intrahepatic transplant andsingle cell suspensions counted then stained for flow cytometry. Forcoinjection experiments, human UCB CD34⁺CD38^(−/lo) (HLA-AZ) cells weredifferentiated on OP9-DL1 cells for 10-12 days, and CD34⁺CD7⁺⁺CD5⁺(proT2) cells were sorted by flow cytometry. On the same day that proT2cells are sorted, CD34⁺CD38^(−/lo) (HLA-A2⁺) cells from umbilicalcord-blood were also sorted. Irradiated (130 cGy) neonatalNOD/SCID/γc^(null) mice from the same litter were injectedintrahepatically with 3.5×10⁴ HSCs alone; 2.5×10⁵ proT2 cells alone, and3.5×10⁴ HSCs together with 2.5×10⁵ proT2 cells.

Flow Cytometry:

Fluorescein isothiocyanate (FITC)-, R-Phycoerythrin (PE)-,allophycocyanin (APC)-, PE-Cy7-, Peridinin chlorophyll protein (PerCP)PerCP-Cy5.5-, Alexa Fluor₇₀₀-, and Alexa Fluor₇₅₀-, conjugatedantibodies were purchased commercially (BD Biosciences or eBioscience).Cell suspensions were FcRII-blocked and stained, and analyzed with anLSR-II cytometer. Data analysis was performed using FlowJo software(Tree Star, Ashland, Oreg.) by gating on live lymphocytes, lack of4′,6-diamidino-2-phenylindole (DAPI) uptake followed by CD45 gating forhuman-specific hematopoietic cells. Numbers in quadrant cornersrepresent percent of gated cells.

Immune Engraftment:

The study of human hematopoiesis employing mouse models first arose inthe late 1980's following the discovery of the scid (sever combinedimmune deficiency) mutation in the C.B-17 mouse strain (Bosma et al.,1983). Such mice harbor a mutation in the prkdc (protein kinase DNAcatalytic protein) gene involved in non-homologous end joining duringTCR and immunoglobulin rearrangement (Bosma et al., 1983), thus, lackingboth mature T and B cells. Soon after, C.B-17 SCID mice were used byMcCune et al (McCune at al., 1988) as an experimental system forstudying human T cell development in relation to HIV-1. Using thismodel, (SCID/hu (thy/liv) model) fragments of human fetal thymus andfetal liver are placed under the kidney capsule of the animal and thegraft is allowed to vascularize. Fetal liver provides a rich source ofhuman HSCs and the fetal thymus provides the environment where the HSCscan differentiate into T cells. Although it was a groundbreaking modelfor studying human lymphocyte development in vivo, most of the engraftedcells were restricted to the fetal explants without seeding the mousebone marrow or other tissues.

Models were then employed to better reflect the ability of humanhematopoietic cells to home and differentiate within the mouseenvironment without human fetal tissues. Many groups were able todemonstrate that sublethally irradiated C.B-17 SCID mice support theengraftment and differentiation of CD34⁺ progenitor cells from humanbone marrow and human cord blood (Lapidot et al., 1992; Vormoor et al.,1994) into multiple hemopoietic lineages. In light of this, CD34⁺ stemcells were coined ‘SCID repopulating cells’ (SRC) as they were capableof repopulating hematopoietic lineages in a SCID mouse. Unfortunately,the levels of engraftment were quite low and T cell development inparticular was typically absent. A major barrier to this engraftment wasinnate immune function still present in the SCID mice. In particular, NKcell function was a critical factor determining host resistance toxeno-engraftment. Use of the non-obese diabetic mouse (NOD) aidedtremendously in facilitating human cell engraftment. The inbred NODmouse strain lacks many aspects of innate immune function due to: (1)complement deficiency due to a mutation in the C5 gene (Baxter andCooke, 1993) (2), compromised NK function and (3) defects in macrophagefunction due to reduced IL-1 secretion. Indeed, introduction of the SCIDmutation onto the NOD background (NOD/SCID) has allowed for successfulhuman engraftment by many groups and is widely used for the study ofhuman hematopoiesis and HSCs (De Smedt et al., 2002; Larochelle et al.,1996). Importantly, Kerre et al demonstrated robust T cell development,albeit in a low percentage of mice, using NOD/SCID animals treated withan antibody blocking murine IL-2Rβ, thus further lowering NK function(Kerre et al., 2002).

Recently, two new mouse models to examine human hematolymphoiddevelopment have emerged: RAG2^(−/−)γ_(c) ^(−/−) and NOD/SCID/γ_(c)^(−/−) immunodeficient mouse strains. Recombinase activation gene 2(RAG2) deficient mice lack RAG function leading to complete abrogationof T and B cell development, due to an absence of TCR and Ig receptorrearrangement. Furthermore, absence of the common cytokine receptor γchain (γ_(c)), a critical subunit for the IL-2, IL-4, IL-7, IL-9, IL-15and IL-21 cytokine receptors, renders these cytokines nonfunctional ontheir target cells. Most importantly, NK cells do not develop in bothRAG2^(−/−)γ_(c) ^(−/−) and NOD/SCID/γ_(c) ^(−/−) mouse strains asIL-15Rγ_(c) is critical for their development (Goldman et al., 1998),thus improving human immune engraftment (Kerre et al., 2002; Legrand etal., 2006; McKenzie et al., 2005). Recently, Traggiai and colleagues(Traggiai et al., 2004) demonstrated that newborn RAG2^(−/−)γ_(c) ^(−/−)transplanted with human CD34⁺ CB cells, developed all major immune cellsubsets. Strikingly, T-lymphopoiesis was supported at high levels, incontrast to the inefficiency of earlier models. The study by Traggiai etal also demonstrated that human T cells can populate the peripheralorgans and elicit anti-viral immune responses indicating that engraftedhuman HSCs differentiate and undergo positive selection events (Traggiaiet al., 2004). Accordingly, it has been suggested that human T cellsundergoing positive selection in the thymus of an immunodeficient mousewould therefore be biased towards mouse MHC molecules and may requiretransplantation of human thymic fragments to observe selection on humanMHC class molecules (Legrand et al., 2006). Alternatively, the type ofAPC used to present viral antigens or the targeted tissue used byinfections agents could determine whether human T cells are positivelyselected on mouse or human MHC molecules. The study by Traggiai et alseems to support the former possibility given that Epstein-Barr Virus(EBV) infects human B cells which can present viral epitopes in thecontext of human MHC molecules (Traggiai et al., 2004). Clearly, in vivomodels with a superior capacity to accept human immune grafts areavailable, rendering them as powerful tools to gain insight into humanhematolymphoid development and to test the safety of in vitro derivedprogenitor T cells in the treatment of immune disorders of the T celllineage.

Results Cellular Analysis of the Sequential Induction of T-CellDevelopment In Vitro.

An important step in the establishment of an effective in vitro systemfor human T-lymphopoiesis is to fully characterize the early stages ofT-cell development. To this end, the inventors performed a temporalkinetic analysis of early developmental changes that occur whenUCB-derived HSCs are induced to differentiate on OP9-DL1 cells. Asexpected, flow cytometric analysis of the starting stem cell populationshowed that sorted CD34⁺CD38^(−/lo) cells do not express markers ofearly T-cell differentiation, such as CD7, CD5, CD1a, and CD10; normarkers of late T-cell differentiation such as CD2, CD4, CD8, and CD3(FIG. 1A).

The inventors made use of CD7 surface expression as a common marker forthe temporal analysis of T-cell differentiation (Barcena et al., 1995;Blom and Spits, 2006). Analysis of CD7 expression in early HSC/OP9-DL1cocultures revealed that this approach recapitulates early and latestages of T-cell development, in which CD7 expression is first detectedat day 4 on CD34⁺ cells, followed by high level expression on CD34⁻cells by days 6-8, and then slightly decreasing on a subset of CD34⁻cells at later time points (beyond day 14) (FIG. 1B).

During the initial week of coculture, as CD34⁺ cells rapidly acquire CD7expression, the overall cell numbers remain constant (FIG. 7) and thecells remain negative for the expression of CD5, CD1a, CD2 and CD4. Byday 8 of culture, CD5 expression is first detected on CD34⁺CD7⁺⁺ cells,which remain CD1a⁻ (FIG. 1B). CD1a⁺ cells begin to be detected by day10, and present on about 15% of the CD7⁺⁺ cells, which correspond tocells that have also started to down-regulate CD34 expression. By day14, expression of CD5 is observed on nearly all of the CD7⁺⁺ cells, withCD1a being expressed on the majority of these cells. Day 14 alsocorresponds to when cells show a blasting phenotype (data not shown) andwhen cellular expansion begins to become apparent (FIG. 7).

At later time points, CD7⁺⁺ and CD7⁺ populations expressing CD2 and CD4begin to predominate (FIG. 1C). In addition, a population of CD7⁺CD1a⁺⁺cells continues to expand, and eventually accounted for nearly 90% ofthe CD7-expressing cells by day 48. In contrast to early time points(days 8-10), in which CD2 expression is low on CD7⁺⁺ cells, by day 48nearly 50% of the cells express high levels of CD2 (FIG. 1C). Theexpression of CD4 on CD7⁺⁺ cells emerges as early as day 12 (FIG. 1B)and continues to increase, eventually accounting for -75% of theCD7-expressing cells by day 48 (FIG. 1C). Although a small percentage ofCD4⁺ cells that lack CD7-expression were detected, the inventors havepreviously reported that these cells belong to the myeloid lineage (LaMotte-Mohs et al., 2005).

Thymus-seeding cells, identified as CD34⁺CD45RA^(hi)CD7⁺, have beenshown to be present in UCB (Haddad et al., 2004) or fetal bone marrow(Haddad et al., 2006). To determine whether a similar population can begenerated in vitro, the inventors looked for cells bearing thisphenotype at early coculture time points. Of note, the starting UCB-HSCpopulation contained a subset of CD34⁺ cells that expressed the CD45RAisoform at low levels (Hao et al., 2001; Payne and Crooks, 2002),however these cells were CDT (FIG. 2A). The analysis showed that CD45RAexpression is up-regulated within the first 4 days on CD34⁺ cells, andby day 6 nearly all CD34⁺CD7⁺⁺ cells express CD45RA (FIG. 2B). Thus, apopulation of CD34⁺CD7⁺⁺CD45RA⁺ cells displaying a thymic-colonizingphenotype, as seen in vivo (Haddad et al., 2004; Haddad et al., 2006),is present in vitro and may also possess thymus reconstitutingpotential.

Molecular Analysis of the Sequential Induction of T-Cell Development inVitro.

Although human HSC/OP9-DL1 cocultures exhibited a cellular expressionpattern consistent with stages of T-cell development observed in thethymus, the precise temporal kinetics of Notch-dependent gene expressionduring early T-cell differentiation (Izon et al., 2002; Radtke et al.,2004) are undefined. The inventors examined the expression of Gata-3,Deltex-1, Rag-1, and Notch-1 transcripts from HSCs cocultured withOP9-control (GFP-only) or OP9-DL1 cells. As shown in FIG. 3A, expressionof Gata-3, Deltex-1, Rag-1 and Notch-1 showed a general trend towardelevated transcript levels in OP9-DL1 as compared to OP9-controlcocultures, with a clear difference starting at around day 14.Consistent with its role in early T-cell specification and commitment(Pai et al., 2003; Rothenberg and Taghon, 2005), Gata-3 expression wasdifferentially induced early and steadily increased over time in OP9-DL1cocultures. Deltex-1, a known Notch-induced target gene (Pear andRadtke, 2003), was also specifically up-regulated as early as day 6 inOP9-DL1 cocultures. Rag-1, an essential gene for TCR gene rearrangements(Shultz et al., 2000), became differentially up-regulated in OP9-DL1cocultures by day 14. Finally, expression of Notch-1 was observedthroughout in both cocultures, but was clearly up-regulated as aconsequence of Delta-like-induced-signaling (Pear and Radtke, 2003).

Although the gene expression kinetics described above are consistentwith the induction of T-lineage differentiation by Notch/Delta-likeinteractions, the inventors sought to more precisely characterize thechanges in gene expression occurring at specific T-cell differentiationstages. To this end, subsets of CD7-expressing cells, with each subsetrepresenting a distinct and sequential stage of T-cell development, wereanalyzed. As shown in FIG. 3B, the developmental progression ofCD7-expressing cells, from a day 40 OP9-DL1 coculture, can be orderedsequentially based on the loss of CD34 and the gain of CD1a expressioninto 4 stages: CD34⁺CD7⁺⁺CD1a⁻, CD34⁻CD7⁺⁺CD1a⁻, CD34⁻CD7⁺⁺CD1a⁺⁺ andfinally CD34⁻CD7⁺CD1a⁺⁺. These subsets, as well as T-cells (CD3⁺) andmyeloid cells (CD33⁺) sorted from UCB as lineage controls, were thenexamined for the expression of Gata-3, Deltex-1, Rag-1, Notch-1, as wellas the myeloid-specific gene Cebpα (Dahl et al., 2003) (FIG. 3C).Up-regulation of Gata-3 transcripts became apparent as CD7⁺⁺ cells loseCD34 surface expression, and remained high during the next stage, but isthen reduced at later stages, which is in keeping with previousobservations (Rothenberg and Taghon, 2005). Deltex-1 was up-regulated ineach of the CD7-expressing subsets when compared to either CD3⁺ matureT-cells or CD33⁺ myeloid cells. Of note, Rag-1 and Notch-1 transcriptup-regulation was most pronounced at the CD34⁻CD7⁺⁺CD1a⁺ stage,consistent with the role of these genes in the generation and functionaloutcomes of the pre-TCR complex (Ciofani et al., 2004). As expected,when CD34 expression is extinguished, CD7-expressing cells become morerestricted to the T-cell lineage, which parallels the observed loss ofCebpa expression within these subsets.

Taken together, human HSC/OP9-DL1 cocultures display stage- andtemporal-specific cellular and molecular signatures, which not onlyrecapitulates key hallmarks of T-lymphopoiesis but also provides asimple and effective way to further dissect the developmental program ofhuman T-cells.

Generation of Functional Human CD8 SP T-Cells from HSCs Cultured withOP9-DL1 Cells.

The inventors have previously reported the ability to generate CD4⁺CD8⁺T-lineage cells from human HSC/OP9-DL1 cocultures (La Motte-Mohs et al.,2005), however whether functional T-cells could be generated was notassessed. To address this, the inventors analyzed long-term cocultures,and FIG. 4A shows the presence of both DP and SP subsets from a day 65coculture. The inventors further examined the CD8 SP subset present inthese cultures for the expression of CD3 and CD27 (Res and Spits, 1999;Vanhecke et al., 1995) typically expressed on mature T-cells. Amongstthe SP CD8 cells (SP8s) found in late cocultures, about 50-60% expressedCD3/αβTCR. Of note, the majority of CD3⁺ SP8s were found to co-expressCD27. In addition, CD27⁺CD3⁺ SP8s were also found to lack CD1aexpression, which is indicative of functional maturity (Res et al.,1997). This is in contrast to CD27⁻CD3⁺ SP8s that continued to expressCD1a, which is characteristic of the preceding stage in T-celldifferentiation (Res et al., 1997).

To address the functional status of in vitro-generated SP8s, theinventors sorted the CD3/TCR-expressing subset (FIG. 4B) and examinedwhether these cells had the capacity to up/down-regulate downstreamdifferentiation markers, proliferate, express cytolyticeffector-function molecules, and secrete γ-interferon (IFNγ) followingstimulation. As shown in FIG. 4C, a blast-like appearance based onforward size scatter is seen in stimulated (S) cells as compared tonon-stimulated (NS) cells. Additionally, stimulated cells up-regulatedCD45RO, CD38 and MHC-class II expression and down-regulated CD27expression, as compared to non-stimulated cells (FIG. 4B). This complexphenotype is unique to activated human T-cells (Holling et al., 2002; Koet al., 1979) and consistent with full effector maturation and greatercytolytic capability (Hamann et al., 1997; van Baarle et al., 2002).Furthermore, to address the extent of cellular proliferation induced byTCR-stimulation, sorted CD3⁺CD8⁺ T-cells were loaded with CFSE. FIG. 4Cshows that stimulated cells undergo many rounds of cell division asindicated by the loss of CFSE compared to non-stimulated cells, withproliferating cells also displaying marked up-regulation of CD25expression.

To determine whether in vitro-derived SP8s can be induced to expresscytotoxic/effector-function molecules, the expression of Granzyme-B andIFNγ were assessed. Intracellular Granzyme-B expression was detected in˜40% of stimulated CD3⁺CD8⁺ T-cells, as compared to non-stimulated cellsthat failed to express Granzyme-B (FIG. 4D). Finally, supernatants fromwells containing in vitro-generated SP8s were analyzed for the presenceof IFNγ following stimulation. As shown in FIG. 4E, supernatants fromstimulated cells showed a significant dose-dependent increase in theamount of IFNγ, as compared to non-stimulated cells.

CD34⁺CD38⁻ and CD34⁺CD38^(lo) UCB Cells Exhibit High T-LymphopoieticPotential.

Several studies have provided evidence that the UCB-CD34⁺ stem cell poolis heterogeneous in terms of its repopulation, differentiation andrenewal potential (Guenechea et al., 2001; Hogan et al., 2002). Indeed,the CD34⁺ population can be subfractionated into distinct subsets basedon CD38 expression (Guenechea et al., 2001; Hogan et al., 2002; Mazurieret al., 2004). The CD38⁻ subfraction contains primitive precursorscapable of long-term reconstitution with slower engraftment kinetics(Hogan et al., 2002). Conversely, UCB cells from the CD34⁺CD38^(lo) orCD38^(+/hi) subsets exhibit different characteristics, giving rise torapid myeloid-erythroid differentiation with short-term repopulatingability (Guenechea et al., 2001; Hogan et al., 2002; Mazurier et al.,2004). However, these studies did not address the frequency ofprogenitors with T-lineage potential among these different CD34⁺subsets. To determine the T-progenitor frequency of the variousUCB-CD34⁺ subsets, CD34⁺CD38⁻, CD34⁺CD38^(lo) and CD34⁺CD38^(+/hi) cellswere sorted (FIG. 8) and placed at limiting cell numbers into wellscontaining OP9-DL1 cells. As shown in Table I, CD34⁺CD38⁻ orCD34⁺CD38^(lo) cells gave rise to T-lineage cells with similaroverlapping frequencies of 1 in 4.8 and 1 in 3.9, respectively, whilethe CD34⁺CD38^(+/hi) subset possessed a nearly 5-fold diminishedT-lineage progenitor frequency of 1 in 19. Thus, the CD34⁺CD38⁻ andCD34⁺CD38^(lo) fraction contains a greater frequency of cells that cangive rise to T-lineage cells.

In Vitro-Generated Pro-T Cells Show Thymus Reconstituting Ability.

The earliest cell thought to colonize the thymus has been described as aCD34⁺ cell expressing CD45RA and CD7 (Haddad et al., 2004; Haddad etal., 2006). The inventors have shown that in HSC/OP9-DL1 coculturescells with this phenotype are present (FIG. 2), however whether thesecells also possess thymus-reconstituting potential remained untested.

To test whether in vitro-generated cells that share a thymus-colonizingcell surface phenotype are able to engraft a thymus, the inventorsutilized a hybrid human/mouse FTOC approach (Fisher et al., 1990).Additionally, the inventors further dissected the CD34⁺CD45RA⁺CD7⁺⁺CD1a⁻progenitor subset based on the presence or absence of CD5 expression,which, as shown in FIG. 5A (and data not shown), CD5 is expressed on˜45% of these cells. To determine whether these T-progenitor subsetshave the potential to engraft and differentiate within a host thymus,CD34⁺CD45RA⁺CD7⁺⁺CD1a⁻ cells that are either CD5⁻ or CD5⁺ (hereafterreferred to as proT1 and proT2, respectively) were sorted from a day 13HSC/OP9-DL1 coculture and placed in FTOCs for 19 days (FIG. 5B).Additionally, the same subsets were placed back onto OP9-DL1 cells (FIG.5C) and their development compared to that occurring in FTOC.

Shown in FIG. 5B, both proT1 and proT2 subsets successfully engraftedthe FTOCs, and their progeny accounted for nearly all of the cellspresent in the lobes, based on human CD45 expression (95%).Additionally, the reconstituted FTOCs contained T-cells that werederived from either the proT1 or proT2 subsets. While the input proT1cells were initially CD34⁺CD7⁺CD5⁻, nearly all of the cells within theengrafted lobes had differentiated into CD34⁻CD5⁺CD1a⁺ T-lineage cells,with 67% also coexpressing CD4 and CD8 and 2-16% expressing either CD8or CD4, with the majority of these being CD4ISPs (data not shown).Similarly, the proT2 cells, which initially expressed CD5, also gaverise to T-cells, however with an increase in the frequency of DP cells(93%). This difference may relate to the later differentiation state ofthe proT2 cells, which as demonstrated in FIG. 21, purified proT1 cellsgave rise to cells with the proT2 phenotype within 24 hours and themajority of these cells reached the next stage by 48 hours.Additionally, purified proT2 cells did not give rise to cells with theproT1 phenotype. The precursor-product relationship from proT1 to proT2is further highlighted by the presence of a small fraction (4%) ofCD34⁺CD7⁺⁺ cells remaining in FTOC seeded with proT1 but not proT2cells. Keeping with this, proT1 cells placed back onto OP9-DL1 cellsalso showed the presence of a CD34⁺CD7⁺⁺ progenitor population, whichwas absent in proT2 cultures (FIG. 5C). Nevertheless, both proT1 andproT2 cells showed a similar overall ability to continue todifferentiate along the T-lineage pathway in these cocultures, givingrise to CD1a⁺ and CD4/CD8-expressing cells.

Since proT1 and proT2 cells share a similar progenitor phenotype withcells found in the human thymus that have been shown to also possessNK-lineage potential, the inventors addressed whether NK cells couldalso be generated from these subsets. Consistent with this, theinventors confirmed that in vitro-derived proT1 and proT2 cells giverise to NK cells when cultured on OP9-control cells, supplemented withIL-15 (FIG. 22). These results are consistent with studies demonstratingthe presence of cells with dual T/NK potential within the CD34⁺CD7⁺⁺thymocyte subset (Sanchez et al., 1994; Spits et al., 1995). Of note,both proT1 and proT2 cells when cultured on OP9-DL1 cells did not giverise to NK cells, rather they continued to differentiate along the Tcell pathway (FIG. 22), which is consistent with the known role of Notchsignaling in maintaining commitment to the T-lineage while inhibitingalternate lineage outcomes. Additionally, methylcellulose assays wereperformed to test the capacity of in vitro generated proT cells to giverise to erythroid, myeloid and granulocytic lineages (Table III). While,sorted CD34+UCB-HSCs generated colonies for all lineages, invitro-generated proT cells displayed a markedly reduced capacity fornon-lymphoid colony formation, including an absence of erythroidpotential from the proT2 cells, further highlighting their diminishedability to generate alternative lineage outcomes with the favoredacquisition of lymphoid potential.

Although both proT1 and proT2 cells can give rise to T-cells, itremained unclear whether these subsets contained a similar progenitorfrequency to reconstitute a host thymus. To address this, sorted proT1and proT2 cells were placed in limiting cell numbers in FTOCs or onOP9-DL1 cells for 7 days, and analyzed by flow cytometry for thepresence of human T-lineage cells. The results shown in Table IIdemonstrate that the proT2 subset displayed a 3-fold higher T-lineageengraftment frequency than that of the proT1 cells (1:400 and 1:1400,respectively). To further examine whether this difference was cellintrinsic, the T-progenitor frequency of these subsets was determined ina limiting dilution assay with OP9-DL1 cells. Of note, and in contrastto the progenitor frequencies observed in FTOCs, the results from thecocultures revealed that both pro-T subsets possess a similar and high(˜1:2) progenitor frequency (Table II).

In light of these findings, it would appear that human pro-T cells,which otherwise display a similarly high T-cell progenitor frequencywhen assayed on the OP9-DL1 monolayer, possess a differential capabilityto engraft a mouse thymus lobe in vitro, which may relate to differencesin the expression of molecules important for entry or niche occupancywithin the thymus. To determine a potential mechanism for the observeddifference in engrafting ability, the inventors analyzed by Q-PCR forthe expression of genes associated with thymus homing or entry (Arroyoet al., 1996; Benz and Bleul, 2005; Goldschneider, 2006; Hirsch et al.,1996; Lai and Kondo, 2007; Rossi et al., 2005; Schwarz et al., 2007).FIG. 6A shows that proT2 cells express higher transcript levels of CCR9(CD199), PSGL-1 (CD162), CD49b (α2 integrin), CD49d (α4 integrin) andCD49e (α5 integrin). A similar trend of elevated expression was observedfor CD29 (β1 integrin) in proT2 cells. Additionally, flow cytometricanalysis of these subsets confirmed that proT2 cells express higherlevels of CD49d than proT1 cells (FIG. 6B). These data are consistentwith previous findings (Arroyo et al., 1996; Hirsch et al., 1996) thatpoint to the CD49d/CD29 heterodimer, which binds to VCAM-1 (CD106)expressed on thymus stromal cells, as well as CCR9 and PSGL-1 asimportant players in facilitating thymus entry by the proT2 cell subset.

In Vitro-Generated Pro-T Cells Injected into Immunodeficient MiceExhibit Thymic Reconstitution Ability In Vivo.

The inventors' finding that human pro-T cells generated in OP9-DL1 cellscould exhibit thymic reconstitution potential in vitro when assayed inFTOC, suggested the possibility that human pro-T cells would similarlydisplay thymic reconstitution potential when assayed in vivo. Toascertain whether in vitro-generated progenitor-T cells can effectivelyreconstitute the T cell compartment in vivo, the inventors have utilizedtwo immunodeficient strains of mice (nonobese diabetic/severe combinedimmunodeficient (NOD/SCIDγ_(c) ^(−/)) (Greiner et al., 1998; Ito et al.,2002; Kollet et al., 2000; Shultz et al., 1995; Vila-Coro et al., 2000)mice and RAG2-deficient, gamma-chain (γ_(c)) deficient (RAG2^(−/−)γ_(c)^(−/−)) (Goldman et al., 1998; Mazurier et al., 1999)) that have beenreported to support the engraftment of human CD34⁺ CB-derived cells(Gimeno et al., 2004; Hogan et al., 1997; Traggiai et al., 2004).

As seen in FIG. 11, RAG2^(−/−)γ_(c) ^(−/) mice injected intrahepaticallywith OP9-DL1 coculture derived-bulk human progenitor-T cells (CD34⁺CD7⁺)displayed human hematopoietic engraftment potential within the thymus asearly as ˜3 weeks post injection as evidenced by the expression of adistinct lymphocytic population that expressed human CD45. Although alymphocytic population could be detected to a lesser degree within thethymus of RAG2^(−/−)γ_(c) ^(−/) mice injected intrahepatically withhuman CD34⁺ stem cells or mock PBS control, these lymphocytes did notexpress human CD45 suggesting these cells were of mouse origin. Uponfurther analysis of RAG2^(−/−)γ_(c) ^(−/) mice injected with bulk pro-Tcells, human CD45⁺-expressing thymocytes displayed a phenotypeconsistent with T cell development (FIGS. 12 and 13). Althoughdifferences in overall cellularity were noted between the twoRAG2^(−/−)γ_(c) ^(−/) mice injected intrahepatically pro-T cells, thevast majority of CD45⁺-gated thymocytes expressed early markers of Tcell differentiation such as CD7, CD5 and CD1a (FIG. 12). Specifically,˜95% of the thymocytes co-expressed CD7 and CD1a, suggesting pro-T cellsefficiently committed to the T cell lineage rather than maintainingtheir input phenotype. Upon closer examination, these CD45⁺-gatedthymocytes also expressed more definitive markers of T celldifferentiation such as CD4, CD8, and CD3 (FIG. 13). By far the vastmajority of these cells exhibited a CD4⁺CD8⁺ double positive (DP)phenotype and could be broken down into CD3-positive and CD3-negativepopulations.

While PBS control injected RAG2^(−/−)γ_(c) ^(−/) mice did notspontaneously generate human CD45⁺ cells in a second experiment, a smallbut detectable population of human CD45⁺ cells were present whenRAG2^(−/−)γ_(c) ^(−/) mice were injected intrahepatically at a higherdose with human CD34⁺ hematopoietic stem cells (FIG. 14). Consistentwith the previous experiment (FIG. 11), a robust population of CD45⁺thymocytes was present in RAG2^(−/−)γ_(c) ^(−/) mice that were injectedwith bulk pro-T cells (FIG. 14). When examined in greater detail (FIG.15), the vast majority of the thymocytes in both the CD34⁺ HSC-injectedand CD34⁴CD7⁺ pro-T-injected RAG2^(−/−)γ_(c) ^(−/) mice expressed bothCD4 and CD8. While single positive CD8 could be detected in thepro-T-injected mouse at three weeks, but not the CD34⁺ HSC-injectedmouse, the vast majority of the single positive cells expressed CD4,suggesting they could be either CD4-intermediate single positive cells(CD4-ISPs) or true CD4-SP cells. When CD45⁺ cells that expressed highlevels of CD3 were examined for the expression, both CD4 and CD8 SPcells could be detected, suggesting that the most of the of CD4 cellspresent within the reconstituted thymus of the RAG2^(−/−)γ_(c) ^(−/)mouse were CD4-ISP and not CD4-SP cells. In contrast to the CD34⁺HSC-injected mouse, which lacked CD4 and CD8 SP cells, thepro-T-injected mouse at three weeks differentiated further and moreefficiently.

The ability of pro-T cells to display thymus reconstitution potentialwas also evaluated in the NOD/SCIDγ_(c) ^(−/) strain of mouse (Greineret al., 1998; Ito et al., 2002; Kollet et al., 2000; Shultz et al.,1995; Vila-Coro et al., 2000). As shown in FIG. 16, NOD/SCIDγ_(c) ^(−/)mice injected intrahepatically with OP9-DL1 coculture derived-bulk humanprogenitor-T cells (CD34⁺CD7⁺), but not CD34⁺ HSCs, expressed humanCD45⁺ cells within their thymus, the vast majority of which, displayedan early T cell phenotype as evidenced by expression of CD5, CD7, andCD1a. Greater than 70% of these developing T cell committed thymocytesexpressed CD4 on their cell surface, while ˜2-20% of the CD4-positivecells co-expressed CD8, suggesting that CD4-ISP cells were transitioningto the DP stage. Taken together pro-T cells are capable of engraftinginto two immunodeficient strains of mice.

Although experiments reconstituting mouse thymus in vitro indicated thatthe proT2 subset displayed a 3-fold higher T-lineage engraftmentfrequency than that of the proT1 cells (1:400 and 1:1400, respectively),it remained to be determined whether similar results would be observedin vivo. Since the inventors demonstrated the ability of bulkCD34⁺⁺CD7⁺⁺ to engraft immunodeficient mice, the inventors tested theability of each proT subset for their threshold for thymusreconstitution in vivo. Cells were sorted and either proT1 or proT2cells were injected into individual neonatal mice at 2.5×10⁴ or 1×10⁴cells, 10-25 fold lower cell numbers, respectively, than used inprevious experiments in which bulk proT cells were used. Three weekspost-injection, the thymuses of mice were harvested and analyzed forengraftment. Results summarized in Table IV show that proT2 cells had ahigher frequency of engraftment than proT1 cells (38% vs 14%) when2.5×10⁴ cells were injected. Furthermore, when only 1×10⁴ cells wereinjected into mice, the inventors observe engraftment of both subsetswith proT2 cells again displaying higher thymus engraftment frequencythan their more immature counterpart.

In Vitro-Generated Pro-T2 Cells Coinjected with Human HSCs intoImmunodeficient Mice Enhance HSC-Derived Thymopoiesis

As shown in FIGS. 14 and 15, proT cells are capable of engrafting andreconstituting the thymuses of RAG2^(−/−)γ_(c) ^(−/−) and NOD/SCIDγ_(c)^(−/)mice. Furthermore, the inventors noted that CD34+ HSCs displayedlower or negligible engraftment capacity, thus the inventors sought todetermine whether the coinjection of in vitro generated proT2 cells withHSCs (derived from a different donor) could positively affect T-lineagereconstitution contributed by HSC-derived cells. To this end, human UCBCD34⁺CD38^(−lo) (HLA-A2⁻) cells were differentiated on OP9-DL1 cells for10-12 days, and CD34⁺CD7⁺⁺CD5⁺ (proT2) cells were sorted by flowcytometry. CD34⁺CD38^(−/lo) (HLA-A2⁺) cells from umbilical cord-bloodwere also sorted. Irradiated (130 rads) neonatal NOD/SCID/γc^(null) micefrom the same litter were divided into 3 groups and injectedintrahepatically with 3.5×10⁴ HSCs, 2.5×10⁵ proT2 cells, or 3.5×10⁴ HSCsmixed with 2.5×10⁵ proT2 cells. At 6 weeks post-injection, the inventorslooked for the presence of human cells in the BM (FIG. 23A) and wereable to trace the origin of the donor cells based on HLA-A2 cell surfaceexpression. The inventors observed the presence of human CD45⁺ HLA-A2⁺ +cells in the BM of mice injected with HSCs only, or receiving both HSCsplus proT2 cells. This population corresponded to cells that weregenerated from HSCs (HLA-A2+), as such, cells with this phenotype werenot observed in mice injected with proT2 cells alone. Further gating onHSC-derived CD45⁺ HLA-A2⁺ revealed that these cells belonged primarilyto the B cell lineage (CD19⁺) with a smaller proportion of these beingmyeloid lineage cells (CD33⁺). Specifically, in the HSC injected mousethese lineages were 85% and 8.5%, respectively, with very similarpercentages observed in both mice coinjected with HSCs and proT2 cells.FIG. 23B shows that human B and myeloid cells derived from HSCs could befound in the spleen. Of note, for both BM and spleen engraftment theinventors did not observe an enhancement or detriment to the HSC derivedlineages when coinjected with proT2 cells; and little to noproT2-derived cells were found at these sites. In contrast, HSC-derivedT-lymphopoiesis was drastically improved by coinjection with invitro-generated proT2 cells. FIG. 23C shows cell surface staining forCD45 and HLA-A2, in which the HSC-only injected mouse showed anextremely low population of human cells in the thymus (as expected basedon the results shown in FIG. 16). However, coinjected mice had adramatic 300-1000 fold increase in the percentage of CD45⁺ HLA-A2⁺(HSC-derived) cells. Additionally, coinjected mice also possessed alarge percentage of cells that corresponded to proT2-derived (CD45⁺HLA-A2⁻) cells (18% and 71%), and as expected, this population was notbe observed in mice that received HSCs only. Further analysis of CD45⁺HLA-A2⁺ and CD45⁺HLA-A2⁻ cells in coinjected mice showed thatHSC-derived cells contained less CD3^(hi) cells (7% and 27%) as well asdecreased percentages of CD4⁺CD8⁺ DP (12% and 58%) T cells as comparedproT2-derived cells, which displayed increased CD3 expression and anincrease in the percentage of DP T cells (over 85% in both mice) (FIG.23D). The delay in their T-lineage differentiation kinetics byHSC-derived cells is consistent with their more immature and primitivestatus at time of injection, as compared to in vitro-generated proT2cells.

Discussion

The early stages of human T-cell development have been broadly-definedby several investigators (Blom and Spits, 2006; Weerkamp et al., 2006c).Here, the inventors have taken advantage of a simple and powerful invitro system to further refine this view by examining thedifferentiation of human UCB-HSCs cultured with OP9-DL1 cells, in whichthe early stages of T-cell development can be readily characterized. Thetemporal kinetic analysis of early and late time points allowed theinventors to discern an ordered pattern of developmental stages, whichis highlighted by the sequential cell surface expression of CD34,CD45RA, CD7, CD5, CD1a, CD2, CD4, CD8, and CD3.

Although the OP9-DL1 system recapitulates the stages of human thymocytedifferentiation, the inventors noted one difference regarding theexpression of CD2, which has been reported to be expressed on some ofthe early CD34⁺ thymocytes as well as in CD34⁺ cells found in the bonemarrow (Haynes and Heinly, 1995; Haynes et al., 1988; Terstappen et al.,1992). The inventors observed CD2 expression only at low levels on cellsthat were down-regulating CD34 expression, and high expression of CD2was seen only at later developmental stages. One possibility for thesedifferences could be an accumulation of CD34⁺ cells with this earlyphenotype within the thymus or that the signals that normally induce theexpression of CD2 on all thymocytes may be lacking in vitro.

Initial findings by the inventors' lab and others have clearly shownthat UCB-CD34⁺ cells can be induced to differentiate to the T-cell fateupon coculture with stromal cells ectopically-expressing DII1 (Jaleco etal., 2001; La Motte-Mohs et al., 2005). However, several groups havedemonstrated that the CD34⁺ population is heterogeneous in regards totheir self-renewal ability, engraftment and lineage potential (Byk etal., 2005; Guenechea et al., 2001; Kollet et al., 2001; Mazurier et al.,2004). With this in mind, the inventors examined whether specific CD34⁺subsets differed in their ability to serve as T-cell progenitors.Additionally, Hogan et al., suggested that the CD34⁺CD38⁻ pool containsa higher frequency of cells with T-cell potential, since NOD/SCID miceengrafted with this fraction showed greater thymus repopulation ascompared to animals receiving CD34⁺CD38^(lo) or CD38^(+/hi) cells (Hoganet al., 2002). In keeping with this, the inventors' results indicatedthat the CD38^(+/hi) fraction has a significantly 5-fold lower T-cellpotential than the more primitive CD38⁻ or CD38^(lo) subsets, whichsurprisingly showed similar T-progenitor frequencies. The comparableprogenitor frequencies by these CD34⁺CD38^(− or lo) cells may beaccounted for by a report suggesting that CD38 is reversibly expressedbetween negative and low levels (McKenzie et al., 2007).

The critical role of Notch signals for inducing T-lymphopoiesis is nowwell-established (Ciofani and Zuniga-Pflucker, 2007; Pear and Radtke,2003). Here, the inventors identified the stages of T-cell developmentin which the induction of Notch target genes are first up-regulated.These stages corresponded to when CD34⁺ cells begin to express CD7 athigh levels, with some further induction following the loss of CD34expression. The inventors' findings are supported by several studiesdemonstrating that UCB-CD34⁺CD7-expressing cells are strongly biased tothe lymphoid lineage with very little myeloid potential (Haddad et al.,2004; Hao et al., 2001; Hoebeke et al., 2007). These observations areconsistent with the notion that T-cell specification occurs early,within the first week, and therefore these Notch-induced CD34⁺CD7⁺⁺cells would likely show an increased T-progenitor frequency. Indeed, theinventors' results indicated that following Notch/Delta-likeinteractions, CD34⁺CD7⁺⁺ cells show a 2-fold higher T-progenitorfrequency than the initial UCB-CD34⁺CDT cells. These findings suggestedthat the HSC/OP9-DL1 cocultures readily support the generation of T-cellprogenitors, which could be akin to thymus-colonizing cells.

It is well-established that the thymus is continuously seeded withblood-borne progenitors, as the thymus-resident progenitors do notpossess self-renewing potential (Donskoy and Goldschneider, 1992). Astudy by Haddad et al. (Haddad et al., 2006) proposed thatthymus-colonizing cells express CD34⁺CD7⁺⁺CD45RA⁺. Cells with a similarphenotype are detected in HSC/OP9-DL1 cocultures, and here the inventorsshow that these cells were able to serve as thymus-colonizing cells.Additionally, the inventors noticed the presence of two distinctprogenitor subsets within the CD34⁺CD7⁺⁺CD1a⁻ population, termed proT1(CD5⁻) and proT2 cells (CD5⁺). Both subsets are capable of thymusreconstitution, however, when used in limiting dilution assays, theinventors observed dramatic differences in their ability to engraft ahost thymus in vitro, with the more mature proT2 cells showing a 3-foldhigher progenitor frequency than proT1 cells. In contrast, when assayedon OP9-DL1 cells both pro-T subsets exhibited statistically similarprogenitor frequencies, which were also dramatically higher (200-600×)than those observed in FTOC. These findings suggest that human pro-Tcells, which otherwise possess high T-cell potential, are affected byxenogeneic barriers present in the mouse FTOC system, which severelylowers their engraftment effectiveness. Additionally, the inventorsnoted that these pro-T subsets differed in the expression of CCR9,PSGL-1 and multiple integrins, which serve to provide a potentialmechanism for the enhanced engrafting ability demonstrated by proT2cells. The higher expression levels of these molecules by proT2 cellswas specific, in that the transcript levels of Cebpa and Gata-2 werehigher in the proT1 subset, which is consistent with their more immaturestatus (FIG. 9).

The HSC/OP9-DL1 cocultures not only serve to characterize progenitorfunction or early events in human T-cell development, but may alsoprovide a simple method for the generation of functional T-cells invitro. This approach may be applicable to cell-based immunotherapiesthat presently capitalize on T-cell effector-function to induce/enhanceanti-tumor eradicating immunity (Rosenberg et al., 2008). Indeed, theinventors now provide clear evidence for the maturation offunctionally-responsive SP8s generated from HSC/OP9-DL1 cocultures. Thisraises the question of which cell-type, within the cultures, ismediating the MHC-dependent positive selection of SP8s. It is unlikelythat the OP9 cells, which express mouse MHC class I that is noteffectively recognized by human CD8 molecules (Irwin et al., 1989),would supply the required positive selection signals. Rather, a humanMHC class I-expressing UCB-derived cell, which may or may not be aT-lineage cell, is likely to be the conveyor of these signals.Additionally, the inventors also noted the appearance of CD3⁺CD4⁺T-cells (FIGS. 4A and 4B), which could be similarly selected by humanMHC class II-expressing cells. However, in contrast to the SP8s, thesecells do not show the hallmarks of functional mature T-cells (data notshown), and may represent transitional cells that require additionaldifferentiation signals that are not readily available in thesecultures.

Human HSC/OP9-DL1 cultures showed robust and continued expansion of DPcells, and could be observed in cultures lasting up to or beyond4-months (FIG. 10). CD4⁺CD8⁺ DP cells are known to be short-lived(Shortman et al., 1990), and thus their presence at these late-timepoints implies that a progenitor cell is maintained and sustains thispopulation. Of note, the inventors are able to detect a population ofCD34⁺ pro-T cells at these late-time points, but it is unclear whetherthese cells persist over time from an initial large pool of progenitorsor from an ability to self-renew and expand in these cultures. Onepossible mechanism would involve Notch signals in the maintenance orextended self-renewal of a progenitor cell subset (Karanu et al., 2000;Karanu et al., 2001; Varnum-Finney et al., 1998), however this notionremains to be directly examined.

Our findings demonstrate that CD34⁺CD7⁺⁺ T-progenitors expressing CD5possess a higher progenitor potential in their ability to engraft FTOCsthan their CD5-negative counterpart. The insight obtained from thisanalysis makes these CD5⁺ proT2 cells an attractive subset for furtherstudies to evaluate their immune reconstitution potential in mousemodels (Legrand et al., 2006). Upon further analysis, bulk CD34⁺CD7⁺⁺T-progenitors derived from human umbilical cord blood HSCs and generatedin vitro utilizing the OP9-DL1 coculture system are capable of thymicreconstitution in two immunodeficient mouse models (FIG. 11-16). Thesethymocytes bear the hallmark signature of committed T-lineage cellsthrough the expression of CD7, CD5, CD1a, CD4 and CD8 on their cellsurface. Although the vast majority of human thymocytes at three weekspost intrahepatic injection of progenitor-T cells are CD4 CD8 doublepositive cells, a small percentage of CD4- and CD8-single positive cellscan be detected amongst gated CD45⁺CD3^(hi) cells. These single positivecells have yet to appear in peripheral organs such as the spleen (datanot shown), suggesting that positive and negative selection initiated bystromal elements within the mouse thymus and/or human antigen presentingcells derived from progenitor-T cells, have yet to occur and thus,premature for thymic export. The inventors' data also demonstrate thathuman CD34+ HSCs also exhibit thymic reconstitution followingintrahepatic injection into immodeficient strains of mice and areconsistent with previous reports by Gimeno et al. and Traggiai, et al.(Gimeno et al., 2004; Traggiai et al., 2004). In the inventors' hands,thymic reconstitution potential from CD34⁺ HSCs appears less efficientand robust than from CD34⁺CD7⁺ bulk progenitor-T cells. Two and notnecessarily mutually exclusive possibilities can be advanced to explainthis observation. In one scenario, while sub-lethal irradiation is notrequired to condition the bone marrow of RAG2^(−/−)γ_(c) ^(−/−) two weekneonates, to accept an HSC cell engraftment Gimeno, 2004 #1087}, it mayfacilitate bone marrow engraftment and the subsequent generation ofthymic colonizing cells reported and detected by Haddad et al in thefetal liver and bone marrow of humans (Haddad et al., 2004; Haddad etal., 2006). It is therefore plausible that should such cells begenerated by human HSC engraftment into immunodeficient mice, that thesecells would colonize and differentiation into human T cell lineage cellswithin the host thymus. Alternatively, thymic colonizing cells might bepresent in limiting numbers within the heterogeneous populations sortedby a CD34⁺CD38^(−/lo) phenotype. In both scenarios, it is conceivablythat human HSCs would exhibit delayed kinetics thymic reconstitutionpotential compared to human bulk progenitor-T cells.

It has been established that patients undergoing autologous orallogeneic hematopoietic stem cell transplants (HSCT) for the treatmentof various hematological disorders display a profound defect in T cellrecovery after HSCT. In contrast to the majority of hematopoietic cellswhose levels are restored within weeks post transplant, T-lineagerecovery is impaired in both cell number and function for up to 2 yearsor it may never recover (Fry and Mackall, 2005). This delay or absenceresults in impaired immune function and is associated with increasedsusceptibility to infection or relapse. Similar to the approachpublished by Van den Brink's group (Zakrzewski et al., 2006a) usingmouse precursors, the inventors' results demonstrate that invitro-generated human proT or proT2 cells dramatically improvedHSC-derived T-lymphopoiesis, which in fact was not typically observed inmice receiving HSCs alone (FIG. 23). Furthermore the coinjection of invitro-generated proT cells with HSCs did not affect HSC-derivedmyelopoiesis or B-lymphopoiesis indicating a targeted effect on thegeneration of T cells from HSCs. A potential mechanism for this effectmay be due to the rapid restoration of the host thymic niches, involvingcell cross talk between T-lineage to thymic stromal cells, resulting inimproved stromal cell cellularity and function, such as cytokine andchemokine production that may then result in enhanced HSC-derivedT-progenitor migration and recruitment from the bone marrow into thethymus. Another explanation, for the observed results may be a direct“piggy-back” of proT cells at the time of injection allowing HSCs tobypass the bone marrow and enter the thymus immediately by attaching tothe proT cells.

Taken together, the inventors' data suggests that rapid immunereconstitution for the treatment of immunodeficiency may be facilitatedthough the utilization of progenitor T cells either in concert or in theabsence of hematopoietic stem cell approaches. Such approaches can betailored or genetically-engineered to generate large numbers of theprogenitor T cells described herein and their progeny to treatimmunodeficiency, triggered by cancer chemo/radio therapeutic regimensand HIV infection, or even restore proper immune function and regulationfor the suppression of autoimmunity.

Indeed, the use of in vitro-derived progenitor T-cells may provetherapeutically relevant over mature effector T-cells by avoiding issuessuch as graft versus host disease, as these cells would undergo positiveand negative selection within the host thymus (Zakrzewski et al.,2006b). It is possible then to speculate that in vitro-generatedT-progenitor cells may eventually serve as a viable option forcell-based therapies (La Motte-Mohs et al., 2007; Zakrzewski et al.,2008), as these cells can be generated in large numbers, allowing fornovel strategies to be developed for the re-establishment ofadaptive-immunity in immunocompromised individuals.

Example 9 Progenitor T Cells are Generated Following Coculture onOP9-DL4 Cells Materials and Methods

Sorted human cord blood derived-HSCs were placed on OP9-control, OP9-DL1or OP9-DL4 cells and cocultured for 24 or 40 days in presence ofrecombinant human cytokines Flt-3L (5 ng/ml) (R&D Systems, Minneapolis,Minn.) and IL-7 (5 ng/ml) (Peprotech, Rocky Hill, N.J.) as previouslydescribed. At day 24 of coculture, developing cells were stained in FACSBuffer (Hank's Balanced Salt Solution (HBSS) 1×—no phenol, no Ca²⁺ noMg²⁺, bovine serum albumin (BSA) 1.0% and sodium azide 0.05%) with thefollowing human antibodies: PE-CD4 [clone RPA-T4], FITC-CD8 [cloneRPA-T8], PE-CD7 [clone M-T701], APC-CD1a [clone HI149], biotin-CD5[clone UCHT2], FITC-TCR-αβ [T10B9.1A-31], FITC TCR-γδ [B1.1], APC-CD3[UCHT2], PE-TCRv_(β)3 [JOVI-3], PE-TCRv_(β)5 [MH3-2], PE-TCRv_(β)8[JR2], PE-TCRv_(β)12 [S511], PE-TCRv_(β)23 [AHUT7] (all purchased fromBD-Pharmigen, San Jose, Calif.), and biotin-pre-Ta (a gift from Dr.Maria Louisa Toribio) against the appropriate isotype controls. Afterincubation, cells were washed and stained with either FITC-Streptavidin(SAv) and APC-SAv secondary reagents (also purchased from BD Pharmigen)for biotin-labeled cells primary antibodies. Following a secondincubation and wash, cells were resuspended in FACS buffer containingpropidium iodide (0.2 μg/ml) and were run on a FACSCalibur(BD-Biosciences) flow cytometer. Data analysis was performed usingFlowJo software (Tree Star, Ashland, Oreg.) by gating on livelymphocytes and lack of propidium iodide uptake. GFP-expressing OP9stromal cells were excluded through GFP expression and side scattergating. This procedure eliminated 99% of contaminating GFP-expressingOP9 stromal cells. Numbers in quadrant corners represent percent ofgated cells.

Results

As OP9-DL1 cells supported robust T cell development generating bothprogenitor T and double positive T cells (FIGS. 17, 18, and 20), theinventors undertook studies to determine whether OP9 cells transduced toexpress the stronger affinity ligand for the Notch1 receptor (Besseyriaset al., 2007), Delta-like-4 (OP9-DL4 cells) could also support thedifferentiation of umbilical cord blood-derived HSCs towards the T celllineage.

As seen in FIG. 17, human HSCs cocultured on OP9-DL1 or OP9-DL4 cells,but not OP9-control cells, generated CD4 CD8 double positive T cellsfollowing 24 days of coculture. Specifically, double positive T cellsaccounted for between ˜15-30% of the lymphocyte population whencocultured on OP9-DL4 or OP9-DL4 cells respectively and proceededthrough the CD4-intermediate single positive stage (ISP). At this stageof T cell development, where double positive T cells are starting toemerge, pre-Ta expression (Carrasco et al., 2002), a key moleculeinvolved in developing T cell survival and expansion, was also evidenton CD4-positive cells and CD4-negative cells in OP9-DL1 and OP9-DL4cocultures, but not OP9-control cocultures. As pre-Ta expression wasobserved on both CD4-positive and CD4-negative cells, the inventorsundertook further studies to determine whether progenitor T cellpopulations that expressed (proT1) or lacked (proT2) CD5 could beevidenced. FIG. 18 shows that OP9-DL1 and OP9-DL4 cocultures, but notOP9-control cocultures, generate cells of the T lineage which can bebroken down into three populations, CD7⁺CD1a⁺⁺ (more mature T cells),CD7⁺⁺CD1a⁺ (committed T cells), and CD7⁺⁺CD1a⁻ (specified progenitor Tcells). Consistent with its role as a T cell marker, CD5 is nearlyubiquitously expressed on the CD7⁺CD1a⁺⁺ and CD7⁺⁺CD1a⁺. In contrast,progenitor CD7⁺⁺CD1a⁻ cells can be broken down into two populations bythe absence or presence of CD5 expression, proT1 cells and proT2 cellsrespectively.

As both OP9-DL1 and OP9-DL4 cocultures generated progenitor T cells andtheir more differentiated progeny, the inventors then undertook studiesto determine whether continued coculture would result in the emergenceof T cells that expressed TCRαβ or TCRγδ. FIG. 19 shows that whileTCRαβ⁻ or TCRγδ⁻ expressing cells can detected amongst the gatedCD7⁺⁺CD1a⁻CD7⁺⁺CD1a⁺, both TCR-bearing subsets are increased in the moremature CD7⁺CD1a⁺. In order to determine whether developing TCRαβutilized different V-beta regions, developing T cells from OP9-control,OP9-DL1, and OP9-DL4 cocultures were stained against CD3 and severalV-beta regions. As FIG. 20 illustrates, developing cells from OP9-DL1cocultures and OP9-DL4 contained CD3-expressing cells (˜38% and ˜13%,respectively) compared to OP9-control cocultures, which lackedCD3-expressing cells. Furthermore, multiple V-beta usage was observed onCD3-expressing cells in OP9-DL1 cocultures, and to a lesser extentOP9-DL4 cocultures, compared to isotype controls. Specifically, of theV-betas that were examined by flow cytometric analysis, V 3 and V 5expression was most readily detected. Taken together, these results showthat OP9-DL4 cells behave similarly to OP9-DL1 cells in their capacityto generate progenitor T cell subsets: proT1 and proT1 cells, that canfurther undergo differentiation to give rise to more mature T cells.

Discussion:

The results showing that OP9-DL4 cells, like OP9-DL1 cells, can generateboth progenitor T and more differentiated T cell progeny, supports thenotion that additional Notch receptor ligands such as Delta-like-4 mayoffer additional signals that may further enhance or promote directeddifferentiation of human HSCs towards cells of the T cell lineage.Whether these Delta-like-4 signals are distinct and/or overlappingremains to be further elucidated and is thus far difficult to testexperimentally due to the lack of commercially available, non-crossreacting, ligand specific-monoclonal antibodies. Recently, it has beendemonstrated that Delta-like-4 is the favored ligand that binds withhigher affinity to the Notch1 receptor suggesting that Delta-like-4 maybe the ligand with the greatest capacity to induce T cell and support Tcell development (Besseyrias et al., 2007). Interestingly, human T celldevelopment induced by OP9-DL1 or OP9-DL4 cocultures seems to suggestthat while OP9-DL4 support T cell development, by far OP9-DL1 cells seemsuperior in their capacity to induce and support robust human T celldevelopment. It should be noted that in the inventors' coculture system,comparable level of Delta-like-expression is difficult to ascertainthrough reporter GFP-expression alone. Thus, given the over-expressionof both Delta-like-1 and Delta-like-4 and their capacity to endocytosis(Bray, 2006), it becomes unclear whether the overall signal strengthtransduced to Notch receptor bearing differentiation cells, masks orexacerbates the differences observed upon supra-optimal expression foundon Delta-like molecules within the OP9-DL1 or OP9-DL4 coculture system.To address this issue, the inventors have begun to engineer taggedversions of OP9-DL1 and OP9-DL4 cells to assess the expression ofprotein levels within these cells to determine whether distinct orsimilar signals are transmitted towards developing progenitor T cells.Nevertheless, it is clear from the inventors' studies that both OP9-DL1and OP9-DL4 cells can support the robust and directed-differentiation ofhuman umbilical cord blood-derived HSCs towards cells of the T lineage,generating large numbers of progenitor T cell subsets, proT1 and proT2as well as their more differentiated progeny.

Example 10 Human Embryonic Stem Cells (hESCs) and Human InducedPluripotent Stem Cells (hiPSCs) Differentiate into Early T-Lineage Cellswhen Cultured with OP9-DL1 or OP9-DL4 Cells Materials and Methods

As previously described (Kennedy et al., 2007) human ESCs are aggregatedto form embryoid bodies (EB) and then sequentially induced todifferentiate towards the hematopoietic lineage through the sequentialaddition of exogenous cytokines. Briefly, during the EB formation,cytokines were added as follows: bone morphogenic protein 4 (BMP4) 10ng/ml at day 0-4, basic fibroblastic growth factor (bFGF) 5 ng/ml at day1-8, Activin A 0.3 ng/ml at day 2-4, vascular growth factor (VEGF) 15ng/ml at day 4-8, dickkopf-1 (Dkk1) 50 ng/ml at day 4-6, interleukin 11(IL-11) 5 ng/ml at day 6-8, IL-6 10 ng/ml at day 6-8, insulin-likegrowth factor IGF-1 25 ng/ml at day 6-8, stem cell factor (SCF) 100ng/ml at day 6-11, thrompoietin (TPO) 50 ng/ml at day 8-11, IL-3 50ng/ml at day 8-11, erythropoietin 4 units at day 8-11, Flt3-L 320 ng/mlat 8-11. Following 9-11 days of EB culture, sorted CD34⁺ and CD34⁻ cellswere seeded onto OP9-DL1 cells (or OP9-DL4), cultured for 20 days, andassayed for T cell potential using flow cytometry. During the OP9-DL1(or OP9-DL4) coculture period, media was changed twice a week, andcocultures were transferred onto new OP9-DL1 (or OP9-DL4) cells. Flt3-L5 ng/ml, IL-7 5 ng/ml, were given during each media change. SCF 100ng/ml was given only during the first 14 days of OP9-DL1 (or OP9-DL4)cocultures.

Human ESC and Human iPSC Differentiation into T-Lineage Cells

Although sustained and continuous T cell development can be derived invitro from UCB-HSCs and can generate CD4⁺CD8⁺ DP, CD4⁺ SP and CD8⁺ SPcells, human embryonic stem cells (hESCs) are an attractive source forgenerating progenitor T cells for future immune-reconstitution studies.Unlike HSCs obtained from other sources, hESCs can be maintained easilyin their undifferentiated state, possess unlimited expansion potential,and are easily malleable for genetic modification. Thus far, thegeneration of T cells from hESCs has remained possible but inefficientrelying on cumbersome methodology and poorly defined inductive events(Galic et al., 2006; Galic et al., 2009; Timmermans et al., 2009). Thisis due in large part to the poor understanding of how hESCsdifferentiate in vitro. Yet, hESCs can differentiate in culture to formall three germ layers (ltskovitz-Eldor et al., 2000; Schuldiner et al.,2000) and there has been limited success in inducing hESCs to developinto hematopoietic cells and B and NK cells (Kaufman et al., 2001; Wollet al., 2005). In particular, there has been only three reports showingthat hESCs can yield T cells in vivo, two of which required the directinjection of human ESC-derived CD34⁺ cells into conjoint humanthymic/liver (Thy/Liv) tissues implanted under the kidney capsule ofsublethally irradiated immunodeficient SCID-mice (Galic et al., 2006;Galic et al., 2009). The third report (published after the initialsubmission of the provisional patent application) although promisingrelied on morphological visualization of hematopoietic zones onOP9-control rather than the isolation of specific specific subsets basedon cellular markers of differentiation, which then had to be excised andpurified onto OP9-DL1 cells (Timmermans et al., 2009). Importantly,there are no reports to date regarding the generation of T lymphocytesfrom human ESCs entirely in vitro—underscoring the need to develop asimple and effective in vitro system for T cell development.

Results

Using the two-stage protocol method for the differentiation of hESCs(Kennedy et al., 2007), sorted CD34⁺⁺ cells, but not CD34⁻ cells, couldgenerate immature early cells of the T-lineage by 20 days of OP9-DL1coculture, as evidence by the expression of CD7 and CD5 (FIG. 24A).Furthermore, the inventors have extended these findings to hiPSCs, alsoobtained from the Keller group, and as shown in FIG. 24B, using asimilar protocol as above, hiPSCs were sorted for CD34⁺⁺ cells and thencultured for 22 days with OP9-DL4 cells. This coculture approach alsogave rise to early T-lineage cells expressing CD7 and CD5, similar tothe cell surface phenotype obtained from UCB-HSC/OP9-DL1 cocultures.

Discussion

The present example shows the ability to generate CD7⁺CD5⁺ humanT-lineage cells, which has not been previously demonstrated using aprospective isolation of hESC or hiPSC-derived CD34⁺ progenitors. Theinventors feel that the defined culture method of EB formation using aspecific cocktail of cytokines followed by culture with OP9-DL1 orOP9-DL4 stromal cells that induce high Notch signaling within thesecells allows for the efficient generation of human T-lineage cells fromthese primitive progenitors.

The ability to readily obtain large numbers of in vitro-generated T cellprogenitors, which can be derived from defined sources of stem cells,whether from UCB-HSCs, hESCs, and hiPSCs, opens new opportunities forthe treatment of T cell immunodeficiencies, acquired or genetic inorigin.

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE I Progenitor frequency analysis of human hematopoietic stem cellsubsets. Progenitor Frequency⁻¹ HSC subset^(a) [95% confidencelimits]^(b) CD34⁺ CD38⁻ 4.76 [3.66-6.21] CD34⁺ CD38^(lo) 3.85[2.94-5.06] CD34⁺ CD38^(+/hi)  19.30 [14.77-25.22] ^(a)CD34⁺ CD38⁻,CD34⁺ CD38^(lo), and CD34⁺ CD38^(+/hi) HSCs were placed in limitingnumbers in wells of a 96-well/plate containing OP9-DL1 cells, andcultured for 11 days before harvesting for flow cytometric analysis.^(b)Individual wells were scored for the presence of T cells based onCD45⁺ CD7⁺⁺ staining. Statistical analysis was performed via the methodof maximum likelihood applied to the Poisson Model (Fazekas de St,1982).

TABLE II Progenitor frequency analysis of progenitor T-cell subsets.Culture Progenitor Frequency⁻¹ Pro-T subset^(a) System^(b) [95%confidence limits]^(c) ProT1 - CD34⁺ 7⁺ 5⁻ FTOC 1384.72 [979-1959]ProT2 - CD34⁺ 7⁺ 5⁺ FTOC 411.74 [256-663] ProT1 - CD34⁺ 7⁺ 5⁻ OP9-DL1   2.52 [1.75-3.63] ProT2 - CD34⁺ 7⁺ 5⁺ OP9-DL1    1.95 [1.35-2.80]^(a)CD34⁺ CD38^(−/lo) UCB-derived cells were cultured on OP9-DL1 cellsfor 12-14 days and proT1/proT2 cells, with the indicated phenotypes,were obtained by flow cytometric cell sorting. ^(b)Pro-T subsets wereplaced in limiting numbers in FTOC or in wells of a 96-well/platecontaining OP9-DL1 cells and cultured for 7 days before harvesting forflow cytometric analysis. ^(c)Individual lobes or wells were scored forthe presence of T cells based on CD45⁺ CD7⁺⁺ or CD7⁺⁺ CD1a^(−/+)staining, respectively. Statistical analysis was performed via themethod of maximum likelihood applied to the Poisson Model (Fazekas deSt, 1982).

TABLE III Assessment of erythroid, mycloid, megakaryocytic andgranulocytic potential of CD34+ UCB cells and various OP9-DL1coculture-derived subsets. CFU-Mix BFU-E CFU-GM CFU-G CFU-M Averagecolony Average colony Average colony Average colony Average colony 500cells plated number (n = 2) number (n = 2) number (n = 2) number (n = 2)number (n = 2) CD34⁺ CB (control) 7.5 66.5 10.5 15 2 CD34⁺CD7⁺CD5⁻CD1a⁻0 11.5 4 6.5 5 proT1 CD34⁺CD7⁺CD5⁺CD1a⁻ 0 0.5 4 5.5 17 proT2 CD34⁻CD7⁺ 00 0 0 0 coculture-derivedThe presence of clonogenic myelo-erythroid progenitors (BFU-E),granulocyte-macrophage colony forming units (CFU-GM), granulocyte colonyforming units (CFU-G), macrophage forming units (CFU-M) andmacrophage-megakaryocyte, erythroid, macrophage, granulocyte (CFU-mix)potential was evaluated by plating 500 sorted in vitro-derived cells(proT1, proT2, and CD34⁻ CD7⁺ subsets) into semi-solid media (1%methylcellulose). CD34⁺ cells sorted from UCB served as a positivecontrol. Colonies were counted from duplicate cultures and the averagenumber of colonies is shown after 22 days. n, the number experimentalreplicates analyzed.

TABLE IV Engraftment potential of proT1 and proT2 subsets injected intoimmunodeficient neonatal mice. Cell number injected Subset % of miceengrafted  1 × 10⁴ ProT1 16% (n = 6) ProT2 50% (n = 2) 2.5 × 10⁴ ProT114% (n = 7) ProT2 38% (n = 8)ProT1 and proT2 cells were sorted from a day 10 coculture and injectedat the indicated cell numbers into immunodeficient mice. Thymuses wereharvested 21-25 days post injection and engraftment was determined bythe presence of human CD45⁺CD7⁺⁺ cells. The percentage of engrafted miceis shown. n, the number of mice analyzed for each treatment group.

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1-18. (canceled) 19: A method of producing a CD34+CD7+CD5+CD1a−progenitor T cell (pro-T cell) comprising: (a) culturing stem cells orprogenitor cells with cells that express a Notch ligand; and (b)determining the presence of CD34+CD7+CD5+CD1a− pro-T cells in theculture. 20: The method of claim 19 wherein the stem cells are selectedfrom hematopoietic stem cells, embryonic stem cells or inducedpluripotent stem cells. 21: The method of claim 20 wherein thehematopoietic stem cell (HSC) is a human HSC. 22: The method of claim 19wherein the cells that express a Notch ligand are stromal cells. 23: Themethod according to claim 19 wherein the Notch ligand is DL1 or DL4. 24:The method of claim 23 wherein the cell expressing the Notch ligand isan OP-9 cell. 25: A method for increasing the number of T cells in amammal comprising administering to a mammal an effective amount of apurified human progenitor T cell having a phenotype CD34+CD7A-CD5+CD1a−,wherein the human progenitor T cell is isolated according to the methodof claim 19, wherein an increase in CD7+CD5+CD1a+ pro-T cells and cellsof a later T-cell development stage is seen. 26: The method according toclaim 25 wherein the mammal has cancer. 27: The method according toclaim 25 wherein the mammal has HIV/AIDS. 28: The method according toclaim 25 wherein the mammal has an autoimmune disease. 29: The methodaccording to claim 25 wherein the progenitor T cell contains aheterologous gene.